DNA encoding galanin GALR2 receptors and uses thereof

ABSTRACT

This invention provides isolated nucleic acids encoding mammalian galanin receptors, isolated galanin receptor proteins, vectors comprising isolated nucleic acid encoding a mammalian galanin receptor, cells comprising such vectors, antibodies directed to a mammalian galanin receptor, nucleic acid probes useful for detecting nucleic acid encoding a mammalian galanin receptor, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding a mammalian galanin receptor, nonhuman transgenic animals which express DNA encoding a normal or a mutant mammalian galanin receptor, as well as methods of determining binding of compounds to mammalian galanin receptors.

[0001] This application is a continuation-in-part in the U.S. of U.S.Ser. No. 08/721,837, filed Sep. 27, 1996, which is acontinuation-in-part of U.S. Ser. No. 08/626,685 and U.S. Ser. No.08/626,046, both filed Apr. 1, 1996, which are continuations-in-part ofU.S. Ser. No. 08/590,494, filed Jan. 24, 1996, the contents of which arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Throughout this application, various references are referred towithin parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citation for these references may be foundat the end of this application, preceding the sequence listing and theclaims.

[0003] The neuropeptide galanin and its receptors hold great promise astargets for the development of novel therapeutic agents. Galanin iswidely distributed throughout the peripheral and central nervous systemsand is associated with the regulation of processes such as somatosensorytransmission, smooth muscle contractility, hormone release, and feeding(for review, see Bartfai et al., 1993). In the periphery galanin isfound in the adrenal medulla, uterus, gastrointestinal tract, dorsalroot ganglia (DRG), and sympathetic neurons. Galanin released fromsympathetic nerve terminals in the pancreas is a potent regulator ofinsulin release in several species (Ahrén and Lindskog, 1992; Boyle etal., 1994), suggesting a potential role for galanin in the etiology ortreatment of diabetes. High levels of galanin are observed in human andrat anterior pituitary where galanin mRNA levels are potentlyupregulated by estrogen (Vrontakis et al., 1987; Kaplan et al., 1988).The presence of galanin in the hypothalamic-pituitary-adrenal axiscoupled with its potent hormonal effects has led to the suggestion thatgalanin may play an integral role in the hormonal response to stress(Bartfai et al., 1993).

[0004] Within the CNS galanin-containing cell bodies are found in thehypothalamus, hippocampus, amygdala, basal forebrain, brainstem nuclei,and spinal cord, with highest concentrations of galanin in thehypothalamus and pituitary (Skofitsch and Jacobowitz, 1985; Bennet etal., 1991; Merchenthaler et al., 1993). The distribution of galaninreceptors in the CNS generally complements that of galanin peptide, withhigh levels of galanin binding observed in the hypothalamus, amygdala,hippocampus, brainstem and dorsal spinal cord (Skofitsch et al., 1986;Merchenthaler et al., 1993; see Bartfai et al., 1993). Accordingly,agents modulating the activity of galanin receptors would have multiplepotential therapeutic applications in the CNS. One of the most importantof these is the regulation of food intake. Galanin injected into theparaventricular nucleus (PVN) of the hypothalamus stimulates feeding insatiated rats (Kyrkouli et al., 1990), an effect which is blocked by thepeptide galanin antagonist M40 (Crawley et al., 1993). In freely feedingrats, PVN injection of galanin preferentially stimulates fat-preferringfeeding (Tempel et al., 1988); importantly, the galanin antagonist M40administered alone decreases overall fat intake (Leibowitz and Kim,1992). These data indicate that specific receptors in the hypothalamusmediate the effects of galanin on feeding behavior, and further suggestthat agents acting at hypothalamic galanin receptors may betherapeutically useful in the treatment of human eating disorders.

[0005] Galanin receptors elsewhere in the CNS may also serve astherapeutic targets. In the spinal cord galanin is released from theterminals of sensory neurons as well as spinal interneurons and appearsto play a role in the regulation of pain threshold (Wiesenfeld-Hallin etal., 1992). Intrathecal galanin potentiates the antinociceptive effectsof morphine in rats and produces analgesia when administered alone(Wiesenfeld-Hallin et al., 1993; Post et al., 1988); galanin receptoragonists may therefore be useful as analgesic agents in the spinal cord.Galanin may also play a role in the development of Alzheimer's disease.In the hippocampus galanin inhibits both the release (Fisone et al.,1987) and efficacy (Palazzi et al., 1988) of acetylcholine, causing animpairment of cognitive functions (Sundström et al., 1988). Autopsysamples from humans afflicted with Alzheimer's disease reveal agalaninergic hyperinnervation of the nucleus basalis (Chan-Palay, 1988),suggesting a role for galanin in the impaired cognition characterizingAlzheimer's disease. Together these data suggest that a galaninantagonist may be effective in ameliorating the symptoms of Alzheimer'sdisease (see Crawley, 1993). This hypothesis is supported by the reportthat intraventricular administration of the peptide galanin antagonistM35 improves cognitive performance in rats (Ögren et al., 1992). Humangalanin receptors thus provide targets for therapeutic intervention inmultiple CNS disorders.

[0006] High-affinity galanin binding sites have been characterized inbrain, spinal cord, pancreatic islets and cell lines, andgastrointestinal smooth muscle in several mammalian species, and allshow similar affinity for ¹²⁵I-porcine galanin (˜0.5-1 nM).Nevertheless, recent in vitro and in vivo pharmacological studies inwhich fragments and analogues of galanin were used suggest the existenceof multiple galanin receptor subtypes. For example, a galanin bindingsite in guinea pig stomach has been reported that exhibits high affinityfor porcine galanin (3-29) (Gu, et al. 1995), which is inactive at CNSgalanin receptors. The chimeric galanin analogue M15 (galantide) acts asantagonist at CNS galanin receptors (Bartfai et al., 1991) but as a fullagonist in gastrointestinal smooth muscle (Gu et al., 1993). Similarly,the galanin-receptor ligand M40 acts as a weak agonist in RINm5Finsulinoma cells and a full antagonist in brain (Bartfai et al, 1993a).The pharmacological profile of galanin receptors in RINm5F cells can befurther distinguished from those in brain by the differential affinitiesof [D-Tyr²]- and [D-Phe²]-galanin analogues (Lagny-Pourmir et al.,1989). The chimeric galanin analogue M35 displaces ¹²⁵I-galanin bindingto RINm5F membranes in a biphasic manner, suggesting the presence ofmultiple galanin receptor subtypes, in this cell line (Gregersen et al.,1993).

[0007] Multiple galanin receptor subtypes may also co-exist within theCNS. Galanin receptors in the dorsal hippocampus exhibit high affinityfor Gal (1-15) but not for Gal (1-29) (Hedlund et al., 1992), suggestingthat endogenous proteolytic processing may release bioactive fragmentsof galanin to act at distinct receptors. The rat pituitary exhibitshigh-affinity binding for ¹²⁵I-Bolton and Hunter (N-terminus)-labeledgalanin (1-29) but not for [¹²⁵I]Tyr²⁶-porcine galanin (Wynick et al.,1993), suggesting that the pituitary galanin receptor is aC-terminus-preferring subtype. Spinal cord galanin binding sites, whilesimilar to those in brain, show an affinity for the chimeric peptideantagonist M35 intermediate between the brain and smooth muscle (Bartfaiet al., 1991), raising the possibility of further heterogeneity.

[0008] A galanin receptor cDNA was recently isolated by expressioncloning from a human Bowes melanoma cell line (Habert-Ortoli et al.,1994). The pharmacological profile exhibited by this receptor is similarto that observed in brain and pancreas, and on that basis the receptorhas been termed GALR1. The cloned human GALR1 receptor binds nativehuman, porcine and rat galanin with ˜1 nM affinity (K_(i) vs.¹²⁵I-galanin) and porcine galanin 1-16 at a slightly lower affinity (˜5nM). Porcine galanin 3-29 does not bind to the receptor. The GALR1receptor appears to couple to inhibition of adenylate cyclase, withhalf-maximal inhibition of forskolin-stimulated cAMP production by 1 nMgalanin, and maximal inhibition occurring at about 1 μM.

[0009] Recently the rat homologue of GALR1 was cloned from the RIN14Bpancreatic cell line (Burgevin, et al., 1995, Parker et al., 1995; Smithet al., in preparation). The pharmacological data reported to date donot suggest substantial differences between the pharmacologic propertiesof the rat and human GALR1 receptors. Localization studies reveal GALR1mRNA in rat hypothalamus, ventral hippocampus, brainstem, and spinalcord (Gustafson et al., 1996), regions consistent with roles for galaninin feeding, cognition, and pain transmission. However, GALR1 appears tobe distinct from the pituitary and hippocampal receptor subtypesdescribed above.

[0010] The indication of multiple galanin receptor subtypes within thebrain underscores the importance of defining galanin receptorheterogeneity at the molecular level in order to develop specifictherapeutic agents for CNS disorders. Pharmacological tools capable ofdistinguishing galanin receptor subtypes in tissue preparations are onlybeginning to appear. Several high-affinity peptide-based galaninantagonists have been developed and are proving useful in probing thefunctions of galanin receptors (see Bartfai et al., 1993), but theirpeptide character precludes practical use as therapeutic agents. Inlight of galanin's multiple neuroendocrine roles, therapeutic agentstargeting a specific disorder must be selective for the appropriatereceptor subtype to minimize side effects.

[0011] Accordingly, the cloning of the entire family of galaninreceptors for use in target-based drug design programs has beenendeavored. The identification of non-peptide agents acting selectivelyonly at specific galanin receptors will be greatly facilitated by thecloning, expression, and characterization of the galanin receptorfamily.

[0012] The isolation by expression cloning of a novel galanin receptorfrom a rat hypothalamic cDNA library, as well as its pharmacologicalcharacterization in a heterologous expression system is now reported.The data provided demonstrate for the first time the existence of a newgalanin receptor subtype, from now on referred to as the GALR2 subtype,or simply, “GALR2.” The cloning of the human homolog of the rat GALR2receptor is also reported. This discovery provides a novel approach,through the use of heterologous expression systems, to develop subtypeselective, high-affinity non-peptide compounds that could serve astherapeutic agents for eating disorders, diabetes, pain, depression,ischemia, and Alzheimer's disease. The presence of both GALR1 and GALR2in rat hypothalamus suggests that multiple galanin receptors may beinvolved in the regulation of feeding. Pathophysiological disordersproposed to be linked to galanin receptor activation include eatingdisorders, diabetes, pain, depression, ischemia, Alzheimer's disease andreproductive disorders. Accordingly, treatment of such disorders may beeffected by the administration of GALR2 receptor-selective compounds.The localization of GALR2 receptors in other parts of the rat brainsuggests that GALR2 receptors may play a role in cognition, analgesia,sensory processing (olfactory, visual), processing of visceralinformation, motor coordination, modulation of dopaminergic activity,neuroendocrine function, sleep disorders, migraine, and anxiety.

SUMMARY OF THE INVENTION

[0013] This invention provides an isolated nucleic acid encoding amammalian GALR2 galanin receptor. This invention also provides anisolated GALR2 receptor protein. This invention further provides DNA,cDNA, genomic DNA, RNA, and mRNA encoding the GALR2 receptor.

[0014] This invention further provides a vector comprising the GALR2receptor. This invention also provides a plasmid which comprises theregulatory elements necessary for expression of GALR2 nucleic acid in amammalian cell operatively linked to a nucleic acid encoding the GALR2receptor so as to permit expression thereof, designated K985 (ATCCAccession No. 97426). This invention also provides a plasmid whichcomprises the regulatory elements necessary for expression of GALR2nucleic acid in a mammalian cell operatively linked to a nucleic acidencoding the GALR2 receptor so as to permit expression thereof,designated BO29 (ATCC Accession No. 97735). This invention providesmammalian cells comprising the above-described plasmid or vector. Thisinvention also provides a membrane preparation isolated from the cells.

[0015] This invention provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorrresponsing to a sequence present within one of the two strands ofthe nucleic acid encoding the GALR2 receptor contained in plasmid K985,plasmid BO29, plasmid BO39 or plasmid K1045. In one embodiment, theGALR2 receptor is the rat GALR2 receptor encoded by the coding sequenceof plasmid K985. In another embodiment, the GALR2 receptor is the humanGALR2 receptor encoded by the coding sequence of plasmid BO29. Thisinvention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence within (a) the nucleic acid sequence shownin FIG. 1 or FIG. 10, or (b) the reverse complement of the nucleic acidsequence shown in FIG. 1 or FIG. 10. This invention further provides anucleic acid probe comprising a nucleic acid molecule of at least 15nucleotides which is complementary to a unique fragment of the sequenceof a nucleic acid molecule encoding a GALR2 receptor. This inventionalso provides a nucleic acid probe comprising a nucleic acid molecule ofat least 15 nucleotides which is complementary to the antisense sequenceof a unique fragment of the sequence of a nucleic acid molecule encodinga GALR2 receptor.

[0016] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to mRNA encoding a GALR2galanin receptor, so as to prevent translation of the mRNA. Thisinvention also provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to the genomic DNA molecule encodinga GALR2 receptor.

[0017] This invention provides an antibody directed to a GALR2 receptor.This invention also provides a monoclonal antibody directed to anepitope of a GALR2 receptor, which epitope is present on the surface ofa cell expressing a GALR2 receptor.

[0018] This invention provides a pharmaceutical composition comprisingan amount of the oligonucleotide effective to reduce activity of a GALR2receptor by passing through a cell membrane and binding specificallywith mRNA encoding a GALR2 receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane. In an embodiment, the oligonucleotide iscoupled to a substance which inactivates mRNA. In another embodiment,the substance which inactivates mRNA is a ribozyme.

[0019] This invention provides a pharmaceutical composition comprisingan amount of an antagonist effective to reduce the activity of a GALR2receptor and a pharmaceutically acceptable carrier.

[0020] This invention provides a pharmaceutical composition comprisingan amount of an agonist effective to increase activity of a GALR2receptor and a pharmaceutically acceptable carrier.

[0021] This invention provides a transgenic nonhuman mammal expressingDNA encoding a GALR2 receptor. This invention provides a transgenicnonhuman mammal comprising a homologous recombination knockout of thenative GALR2 receptor. This invention provides a transgenic nonhumanmammal whose genome comprises antisense DNA complementary to DNAencoding a GALR2 receptor so placed as to be transcribed into antisensemRNA which is complementary to mRNA encoding a GALR2 receptor and whichhybridizes to mRNA encoding a GALR2 receptor thereby reducing itstranslation.

[0022] This invention also provides a process for determining whether acompound can specifically bind to a GALR2 receptor which comprisescontacting a cell transfected with and expressing DNA encoding the GALR2receptor with the compound under conditions permitting binding ofcompounds to such receptor, and detecting the presence of any suchcompound specifically bound to the GALR2 receptor, so as to therebydetermine whether the ligand specifically binds to the GALR2 receptor.

[0023] This invention provides a process for determining whether acompound can specifically bind to a GALR2 receptor which comprisespreparing a cell extract from cells transfected with and expressing DNAencoding the GALR2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the compound underconditions permitting binding of compounds to such receptor, anddetecting the presence of the compound specifically bound to the GALR2receptor, so as to thereby determine whether the compound specificallybinds to the GALR2 receptor.

[0024] In one embodiment, the GALR2 receptor is a mammalian GALR2receptor. In another embodiment, the GALR2 receptor is a rat GALR2receptor. In still another embodiment, the GALR2 receptor hassubstantially the same amino acid sequence encoded by the plasmid K985.In another embodiment, the GALR2 receptor is a human GALR2 receptor. Instill another embodiment, the GALR2 receptor has substantially the sameamino acid sequence as the sequence encoded by plasmid BO29.

[0025] This invention provides a process for determining whether acompound is a GALR2 receptor agonist which comprises contacting a celltransfected with and expressing DNA encoding the GALR2 receptor with thecompound under conditions permitting the activation of the GALR2receptor, and detecting an increase in GALR2 receptor activity, so as tothereby determine whether the compound is a GALR2 receptor agonist.

[0026] This invention provides a process for determining whether acompound is a GALR2 receptor antagonist which comprises contacting acell transfected with and expressing DNA encoding the GALR2 receptorwith the compound in the presence of a known GALR2 receptor agonist,such as galanin, under conditions permitting the activation of the GALR2receptor, and detecting a decrease in GALR2 receptor activity, so as tothereby determine whether the compound is a GALR2 receptor antagonist.

[0027] This invention provides a compound determined by theabove-described processes. In one embodiment of the above-describedprocesses, the compound is not previously known. In another embodiment,the compound is not previously known to bind to a GALR2 receptor.

[0028] This invention provides a process of screening a plurality ofchemical compounds not known to bind to a GALR2 receptor to identify acompound which specifically binds to the GALR2 receptor, which comprises(a) contacting cells transfected with and expressing DNA encoding theGALR2 receptor with a compound known to bind specifically to the GALR2receptor; (b) contacting the preparation of step (a) with the pluralityof compounds not known to bind specifically to the GALR2 receptor, underconditions permitting binding of compounds known to bind the GALR2receptor; (c) determining whether the binding of the compound known tobind to the GALR2 receptor is reduced in the presence of the compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to theGALR2 receptor of each compound included in the plurality of compounds,so as to thereby identify the compound which specifically binds to theGALR2 receptor.

[0029] This invention provides a method of screening a plurality ofchemical compounds not known to activate a GALR2 receptor to identify acompound which activates the GALR2 receptor which comprises (a)contacting cells transfected with and expressing the GALR2 receptor withthe plurality of compounds not known to activate the GALR2 receptor,under conditions permitting activation of the GALR2 receptor; (b)determining whether the activity of the GALR2 receptor is increased inthe presence of the compounds; and if so (c) separately determiningwhether the activation of the GALR2 receptor is increased by eachcompound included in the plurality of compounds, so as to therebyidentify the compound which activates the GALR2 receptor.

[0030] This invention provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a GALR2receptor to identify a compound which inhibits the activation of theGALR2 receptor, which comprises (a) preparing a cell extract from cellstransfected with and expressing DNA encoding the GALR2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with the plurality of compounds in the presence of aknown GALR2 receptor agonist, under conditions permitting activation ofthe GALR2 receptor; (b) determining whether the activation of the GALR2receptor is reduced in the presence of the plurality of compounds,relative to the activation of the GALR2 receptor in the absence of theplurality of compounds; and if so (c) separately determining theinhibition of activation of the GALR2 receptor for each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which inhibits the activation of the GALR2 receptor.

[0031] This invention provides a method of detecting expression of aGALR2 receptor by detecting the presence of mRNA coding for the GALR2receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with the above-described nucleic acidprobe under hybridizing conditions, detecting the presence of mRNAhybridized to the probe, and thereby detecting the expression of theGALR2 receptor by the cell.

[0032] This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of aGALR2 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition effective todecrease the activity of the GALR2 receptor in the subject, therebytreating the abnormality in the subject. In an embodiment, theabnormality is obesity. In another embodiment, the abnormality isbulimia.

[0033] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by the activation of aGALR2 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition effective toactivate the GALR2 receptor in the subject. In an embodiment, theabnormal condition is anorexia.

[0034] This invention provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific human GALR2receptor allele which comprises: (a) obtaining DNA of subjects sufferingfrom the disorder; (b) performing a restriction digest of the DNA with apanel of restriction enzymes; (c) electrophoretically separating theresulting DNA fragments on a sizing gel; (d) contacting the resultinggel with a nucleic acid probe capable of specifically hybridizing with aunique sequence included within the sequence of a nucleic acid moleculeencoding a human GALR2 receptor and labelled with a detectable marker;(e) detecting labelled bands which have hybridized to DNA encoding ahuman GALR2 receptor labelled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps a-e; and (g)comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

[0035] This invention provides a method of modifying feeding behavior ofa subject which comprises administering to the subject an amount of acompound which is a galanin receptor agonist or antagonist effective toincrease or decrease the consumption of food by the subject so as tothereby modify feeding behavior of the subject. In an embodiment, thecompound is a GALR2 receptor antagonist and the amount is effective todecrease the consumption of food by the subject. In another embodimentthe compound is administered in combination with food.

[0036] In yet another embodiment the compound is a GALR2 receptoragonist and the amount is effective to increase the consumption of foodby the subject. In a still further embodiment, the compound isadministered in combination with food. In other embodiments the subjectis a vertebrate, a mammal, a human or a canine.

BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1 Nucleotide coding sequence of the rat hypothalamic galaninGALR2 receptor (Seq. I.D. No. 7), with partial 5′ and 3′ untranslatedsequences. Start (ATG) and stop (TAA) codons are underlined.

[0038]FIG. 2 Deduced amino acid sequence of the rat hypothalamic galaninGALR2 receptor encoded by the nucleotide sequence shown in FIG. 1 (Seq.I.D. No. 8).

[0039] FIGS. 3A-3C 3A. Diagram of the intron-exon arrangement of the ratGALR2 receptor cDNA contained in plasmid K985. Untranslated regions areindicated by dark hatched segments, and coding region is unmarked exceptfor light gray hatched segments indicating the location of thetransmembrane domains of the rat GALR2 receptor. The black segmentindicates the location of the intron. 3B. Splice junction sequences ofthe rat GALR2 receptor. Nucleotide number 1 is located 45 nucleotidesupstream of the start codon (Seq. I.D. No. 9). 3C. Intron sequence ofrat GALR2 receptor cDNA contained in plasmid K985. Nucleotide number 1is located 45 nucleotides upstream of the start codon (Seq. I.D. No. 9).

[0040] FIGS. 4A-4C Localization of [¹²⁵I]galanin binding sites in ratCNS. FIGS. 4A-1 and 4A-4: Distribution of total [¹²⁵I]galanin binding incoronal sections through the hypothalamus and amygdala. FIGS. 4A-2 and4A-5: Binding which remains in these areas following incubation with 60nM [D-Trp²]galanin₍₁₋₂₉₎. FIGS. 4A-3 and 4A-6: Binding obtained afterincubation with 5 μM porcine galanin, which represents the non-specificbinding condition. FIG. 4B: FIGS. 4B-1 to 4B8: Higher magnificationphotomicrographs of the [¹²⁵I]galanin binding sites in the hypothalamusand amygdala. FIG. 4B-1: Total binding in the paraventricularhypothalamic nucleus (PVN), virtually all of which is removed by 60 nM[D-Trp²]galanin₍₁₋₂₉₎(panel 3B). FIGS. 4B-3 and 4B-4: Binding in theventromedial hypothalamus (VMH), lateral hypothalamus (LH), and zonaincerta (ZI). In these regions, some [¹²⁵I]galanin binding remains afterincubation with 60 nM [D-Trp²]galanin₍₁₋₂₉₎ (FIG. 4B-4). FIGS. 4B-5 and4B-7: Total binding in the amygdala. After incubation with 60 nM[D-Trp²]galanin₍₁₋₂₉₎ (panels 5B and 6B), the binding is markedlyreduced in the piriform cortex (Pir), and to a lesser extent in themedial nucleus (Me), and central nucleus (Ce). However, the binding inthe nucleus of the lateral olfactory tract (LOT) is largely unaffected.FIG. 4C: Panels 4C-1 to 4C-6: Distribution of [¹²⁵I]galanin bindingsites in the anterior forebrain (panel 7) and in the midbrain (panel 8).In the lateral septum (LS) and insular cortex (CTX), much of the totalbinding (panel 7A) is removed by 60 nM [D-Trp²]galanin₍₁₋₂₉₎ (panel 7B).Similarly, the total binding observed in the superior colliculus (SC),central gray (CG), and pontine reticular nucleus (PnO) (panel 8A) ismarkedly diminished (panel 8B). FIGS. 4C-3 and 4C-6: Nonspecific bindingobserved in adjacent sections through the septum and midbrain. Arc,arcuate nucleus;. Ce, central amygdaloid nucleus; CL, centrolateralthalamic nucleus; LOT, nucleus of the lateral olfactory tract; Me,medial amygdaloid nucleus; Pir, piriform cortex; PVN, paraventricularhypothalamic nucleus; SO, supraoptic nucleus; st, stria terminalis; VMH,ventromedial hypothalamic nucleus; ZI, zona incerta.

[0041]FIG. 5. Reverse transcriptase PCR (RT-PCR) of rat GALR2 receptormRNA from various brain regions. The blot was hybridized at highstringency with an oligonucleotide probe corresponding to a portion ofthe predicted V/VI loop of GALR2. Positive controls are indicated by +'sand represent plasmids containing the indicated inserts. Size standardsare indicated at the left in kilobases. Note the additional hybridizingbands intermediate in size between the intron-containing and theintronless product.

[0042] FIGS. 6A-6B. Northern blot analysis of GALR2 receptor mRNA fromvarious rat brain regions. 6A. A Northern blot containing poly A⁺ RNA(˜5 μg) from six different rat brain regions was hybridized at highstringency with a randomly primed radiolabeled fragment representing theentire rat GALR2 coding region (not including the intron). Theautoradiogram represents a four day exposure and reveals a ˜1.8-2.0 kbtranscript. 6B. The blot was reprobed with 1B15 (˜1 kb) to confirm thatsimilar amounts of RNA were present in each lane.

[0043] FIGS. 7A-7B. Northern blot analysis of GALR2 receptor mRNA fromvarious rat tissues. 7A. A Northern blot containing poly A⁺ RNA (˜2 μg)from eight different rat tissues was hybridized at high stringency witha randomly primed radiolabeled fragment representing the entire ratGALR2 coding region (not including the intron). The autoradiogramrepresents a four day exposure and reveals a single ˜1.8-2.0 kbtranscript. 7B. The Northern blot was reprobed for 1B15 (˜1 kb) toconfirm that similar amounts of RNA were present in each lane. A secondNorthern blot (not shown) was also hybridized under the same conditionsand showed similar results (Table 3).

[0044] FIGS. 8A-8D. Rat GALR2 receptor autoradiography in COS-7 cellstransfected with GALR1 and GALR2 cDNAs. ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ wastested as a selective radioligand for GALR2. Panels represent dark-fieldphotomicrographs (200×) of photoemulsion-dipped slides. 8A: Binding of 3nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ to COS-7 cells transiently transfectedwith GALR2. Note positive binding to cells. 8B: Nonspecific binding of 6nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ in the presence of 300 nM porcinegalanin₍₁₋₂₉₎ to COS-7 cells transiently transfected with GALR2. 8C:Binding of 6 nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎ to COS-7 cells transientlytransfected with GALR1. Note absence of binding to cell profiles; smallaccumulations of silver grains represent nonspecific nuclearassociation. 8D: Nonspecific binding of 6 nM ¹²⁵I-[D-Trp²]Galanin₍₁₋₂₉₎in the presence of 600 nM porcine galanin₍₁₋₂₉₎ to COS-7 cellstransiently transfected with GALR1.

[0045] FIGS. 9A-9B. Functional response mediated by LM(tk−) cells stablytransfected with the cDNA encoding the rat GALR2 receptor. 9A:Inhibition of cyclic AMP formation: cells were incubated with varyingconcentrations of porcine galanin₍₁₋₂₉₎, and 10 μM forskolin for 15 min.at 37° C. Data was normalized taking as 0% the basal levels of cyclicAMP (0.06±0.02 pmol/ml) and 100% the cAMP levels produced by forskolinin the absence of agonist (0.26±0.03 pmol/ml). Data is shown asmean±standard error of the mean of four independent experiments. 9B:Phosphoinositide metabolism: cells were incubated for 18 hours in thepresence of 0.5 μCi [³H]myo-inositol. Eleven different concentrations ofporcine galanin₍₁₋₂₉₎ were added in the presence on 10 mM LiCl. Cellswere incubated for 1 hour at 37° C., and [³H]inositol phosphates wereisolated and measured.

[0046]FIG. 10. Nucleotide coding sequence of the human galanin GALR2receptor (Seq. I.D. No. 29), with partial 5′ and 3′ untranslatedsequences. Start (ATG) and stop (TGA) codons are underlined.

[0047]FIG. 11. Deduced amino acid sequence of the human galanin GALR2receptor encoded by the nucleotide sequence shown in FIG. 10 (Seq. I.D.No. 30).

[0048] FIGS. 12A-12C. 12A. Diagram of the intron-exon arrangement of thehuman GALR2 receptor cDNA contained in plasmid BO29. Untranslatedregions are indicated by dark hatched segments, and coding region isunmarked except for light gray hatched segments indicating the locationof the transmembrane domains of the human GALR2 receptor. The blacksegment indicates the location of the intron. 12D. Splice junctionsequences of the human GALR2 receptor. 12C. Intron sequence of humanGALR2 receptor cDNA contained in plasmid BO29 (Seq. I.D. No. 31).

[0049]FIG. 13. Current response in an oocyte injected with 50 pg ofGALR2 mRNA. Holding potential was −80 mV.

[0050]FIG. 14. Autoradiograph demonstrating hybridization ofradiolabeled rGalR2 probe to RNA extracted from rat. The lower band(arrow) represents mRNA coding for the rat GALR2 extracted from tissueindicated at the bottom of the gel. RNA coding for the rat GALR2 ispresent in: the heart, kidney, hypothalamus, hippocampus, amygdala,spinal cord, and cerebellum. mRNA coding for the rat GALR2 was notdetected in RNA extracted from striated muscle or liver.

[0051] FIGS. 15A-15D. Amino acid sequence alignment of the rat GALR2receptor (top row) (Seq. ID No. 8), human GALR2 receptor (middle row)(Seq. ID No. 29) and rat GALR1 receptor (bottom row) (Seq. ID No. 32).Transmembrane domains (TM 1-7) are indicated by brackets above thesequence.

DETAILED DESCRIPTION OF THE INVENTION

[0052] Throughout this application, the following standard abbreviationsare used to indicate specific nucleotide bases:

[0053] C=cytosine

[0054] T=thymine

[0055] A=adenine

[0056] G=guanine

[0057] Furthermore, the term “agonist” is used throughout thisapplication to indicate any peptide or non-peptidyl compound whichincreases the activity of any of the receptors of the subject invention.The term “antagonist” is used throughout this application to indicateany peptide or non-peptidyl compound which decreases the activity of anyof the receptors of the subject invention.

[0058] The activity of a G-protein coupled receptor such as a galaninreceptor may be measured using any of a variety of functional assays inwhich activation of the receptor in question results in an observablechange in the level of some second messenger system, including but notlimited to adenylate cyclase, calcium mobilization, arachidonic acidrelease, ion channel activity, inositol phospholipid hydrolysis orguanylyl cyclase. Heterologous expression systems utilizing appropriatehost cells to express the nucleic acid of the subject invention are usedto obtain the desired second messenger coupling. Receptor activity mayalso be assayed in an oocyte expression system.

[0059] This invention provides an isolated nucleic acid encoding a GALR2galanin receptor. In an embodiment, the galanin receptor is a vertebrateor a mammalian GALR2 receptor. In another embodiment, the galaninreceptor is a rat GALR2 receptor. In another embodiment, the galaninreceptor is a human GALR2 receptor. In an embodiment, the isolatednucleic acid encodes a receptor characterized by an amino acid sequencein the transmembrane region, which has a homology of 60% or higher tothe amino acid sequence in the transmembrane region of the rat galaninGALR2 receptor and a homology of less than 60% to the amino acidsequence in the transmembrane region of any GALR1 receptor. In oneembodiment, the GALR2 receptor is a rat GALR2 receptor. In anotherembodiment, the GALR2 receptor is a human GALR2 receptor.

[0060] This invention provides an isolated nucleic acid encoding a GALR2receptor having substantially the same amino acid sequence as shown inFIG. 2. In one embodiment, the nucleic acid is DNA. This inventionfurther provides an isolated nucleic acid encoding a rat GALR2 receptorhaving the amino acid sequence shown in FIG. 2. In another embodiment,the nucleic acid comprises at least an intron. In yet anotherembodiment, the intron comprises a fragment of the intron sequence shownin FIG. 3C (Seq. I.D. No. 9). In still another embodiment, the nucleicacid comprises alternately spliced nucleic acid transcribed from thenucleic acid contained in plasmid K985. In one embodiment, thealternately spliced nucleic acid is mRNA transcribed from DNA encoding agalanin receptor.

[0061] In one embodiment, the GALR2 receptor has substantially the sameamino acid sequence as the amino acid sequence encoded by plasmid K985(ATCC Accession No. 97426). In another embodiment, the GALR2 receptorhas the amino acid sequence encoded by the plasmid K985. In stillanother embodiment, the GALR2 receptor has substantially the same aminoacid sequence as the amino acid sequence encoded by the plasmid K1045.In yet another embodiment, the GALR2 receptor has the amino acidsequence encoded by the plasmid K1045. Plasmid K1045 comprises anintronless cDNA encoding the rat GALR2 receptor. Plasmid K1045 isfurther characterized by its lack of native 5′ or 3′ untranslatedsequences, such that the plasmid contains only the regulatory elementsnecessary for expression in mammalian cells (e.g., Kozak consensussequence) and the coding sequence of the GALR2 receptor.

[0062] In one embodiment, the human GALR2 receptor has substantially thesame amino acid sequence as the amino acid sequence encoded by plasmidBO29 (ATCC Accession No. 97735). In yet another embodiment, the humanGALR2 receptor has the amino acid sequence encoded by the plasmid BO29.In another embodiment, the nucleic acid encoding the human GALR2receptor comprises an intron. In still another embodiment, the nucleicacid encoding the human GALR2 receptor comprises alternately splicednucleic acid transcribed from the nucleic acid contained in plasmidBO29. In still another embodiment, the human GALR2 receptor hassubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid BO39 (ATCC Accession No. ______). In anotherembodiment, the human GALR2 receptor has the amino acid sequence encodedby the plasmid BO39. Plasmid BO39 comprises an intronless cDNA encodingthe human GALR2 receptor. This invention provides an isolated nucleicacid encoding a GALR2 receptor having substantially the same amino acidsequence as shown in FIG. 11 (Seq. I.D. No. 30). In one embodiment, thenucleic acid is DNA. This invention further provides an isolated nucleicacid encoding a human GALR2 receptor having the amino acid sequenceshown in FIG. 11.

[0063] The observation that both the human and rat GALR2 cDNAs containat least one intron raises the possibility that additional introns couldexist in coding or non-coding regions. In addition, spliced form(s) ofmRNA may encode additional amino acids either upstream of the currentlydefined starting methionine or within the coding region. Further, theexistence and use of alternative exons is possible, whereby the mRNA mayencode different amino acids within the region comprising the exon. Inaddition, single amino acid substitutions may arise via the mechanism ofRNA editing such that the amino acid sequence of the expressed proteinis different than that encoded by the original gene (Burns et al., 1996;Chu et al., 1996). Such variants may exhibit pharmacologic propertiesdiffering from the receptor encoded by the original gene.

[0064] This invention provides a splice variant of the GALR2 receptorsdisclosed herein. This invention further provides for alternatetranslation initiation sites and alternately spliced or edited variantsof nucleic acids encoding rat and human GALR2 receptors.

[0065] This invention provides the above-described isolated nucleicacid, wherein the nucleic acid is DNA. In an embodiment, the DNA iscDNA. In another embodiment, the DNA is genomic DNA. In still anotherembodiment, the nucleic acid molecule is RNA. Methods for production andmanipulation of nucleic acid molecules are well known in the art.

[0066] In another embodiment, the nucleic acid encodes a vertebrateGALR2 receptor. In a separate embodiment, the nucleic acid encodes amammalian GALR2 receptor. In another embodiment, the nucleic acidencodes a rat GALR2 receptor. In still another embodiment, the nucleicacid encodes a human GALR2 receptor.

[0067] This invention further provides nucleic acid which is degeneratewith respect to the DNA comprising the coding sequence of the plasmidK985. This invention also provides nucleic acid which is degenerate withrespect to the DNA comprising the coding sequence of the plasmid K1045.This invention further provides nucleic acid which is degenerate withrespect to any DNA encoding a GALR2 receptor. In one embodiment, thenucleic acid comprises a nucleotide sequence which is degenerate withrespect to the nucleotide sequence described in FIG. 1 (Seq. I.D. No.1), that is, a nucleotide sequence which is translated into the sameamino acid sequence. In another embodiment, the nucleic acid comprises anucleotide sequence which is degenerate with respect to the nucleotidesequence described in Seq. I.D. No. 9.

[0068] In yet another embodiment, this invention further providesnucleic acid which is degenerate with respect to the DNA comprising thecoding sequence of plasmid BO29. In an embodiment, the nucleic acidcomprises a nucleotide sequence which is degenerate with respect to thenucleotide sequence described in FIG. 10 (Seq. I.D. No. 29), that is, anucleotide sequence which is translated into the same amino acidsequence. This invention also provides nucleic acid which is degeneratewith respect to the DNA comprising the coding sequence of the plasmidBO39.

[0069] This invention also encompasses DNAs and cDNAs which encode aminoacid sequences which differ from those of the GALR2 galanin receptor,but which should not produce phenotypic changes. Alternatively, thisinvention also encompasses DNAs, cDNAs, and RNAs which hybridize to theDNA, cDNA, and RNA of the subject invention. Hybridization methods arewell known to those of skill in the art.

[0070] The nucleic acids of the subject invention also include nucleicacid molecules coding for polypeptide analogs, fragments or derivativesof antigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

[0071] G-protein coupled receptors such as the GALR2 receptors of thepresent invention are characterized by the ability of an agonist topromote the formation of a high-affinity ternary complex between theagonist, the receptor, and an intracellular G-protein. This complex isformed in the presence of physiological concentrations of GTP, andresults in the dissociation of the alpha subunit of the G protein fromthe beta and gamma subunits of the G protein, which further results in afunctional response, i.e., activation of downstream effectors such asadenylyl cyclase or phospholipase C. This high-affinity complex istransient even in the presence of GTP, so that if the complex isdestablized, the affinity of the receptor for agonists is reduced. Thus,if a receptor is not optimally coupled to G protein under the conditionsof an assay, an agonist will bind to the receptor with low affinity. Incontrast, the affinity of the receptor for an antagonist is normally notsignificantly affected by the presence or absence of G protein.Functional assays may be used to determine whether a compound binds tothe receptor, but may be more time-consuming or difficult to performthan a binding assay. Therefore, it may desirable to produce a receptorwhich will bind to agonists with high affinity in a binding assay.Examples of modified receptors which bind agonists with high affinityare disclosed in WO 96/14331, which describes neuropeptide Y receptorsmodified in the third intracellular domain. The modifications mayinclude deletions of 6-13 amino acids in the third intracellular loop.Such deletions preferaby end immediately before the polar or chargedresidue at the beginning of helix six. In one embodiment, the deletedamino acids are at the carboxy terminal portion of the thirdintracellular domain. Such modified receptors may be produced usingmethods well-known in the art such as site-directed mutagenesis orrecombinant techniques using restriction enzymes.

[0072] This invention provides an isolated nucleic acid encoding amodified GALR2 receptor, which differs from a GALR2 receptor by havingan amino acid(s) deletion, replacement or addition in the thirdintracellular domain. In one embodiment, the modified GALR2 receptordiffers by having a deletion in the third intracellular domain. Inanother embodiment, the modified GALR2 receptor differs by having anamino acid replacement or addition to the third intracellular domain.

[0073] The modified receptors of this invention may be transfected intocells either transiently or stably using methods well-known in the art,examples of which are disclosed herein. This invention also provides forbinding assays using the modified receptors, in which the receptor isexpressed either transiently or in stable cell lines. This inventionfurther provides for a compound identified using a modified receptor ina binding assay such as the binding assays described herein.

[0074] The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

[0075] This invention also provides an isolated galanin GALR2 receptorprotein. In one embodiment, the GALR2 receptor protein has the same orsubstantially the same amino acid sequence as the amino acid sequenceencoded by plasmid K985. In another embodiment, the GALR2 receptorprotein has the same or substantially the same amino acid sequence asthe amino acid sequence encoded by plasmid K1045. In one embodiment, theGALR2 receptor protein has the same or substantially the same amino acidsequence as shown in FIG. 2. In another embodiment, the GALR2 receptorhas the amino acid sequence shown in FIG. 2. In still anotherembodiment, the GALR2 receptor protein has the same or substantially thesame amino acid sequence as the amino acid sequence encoded by plasmidBO29. In still another embodiment, the GALR2 receptor protein has thesame or substantially the same amino acid sequence as the amino acidsequence encoded by plasmid BO39. In an embodiment, the GALR2 receptorprotein has the same or substantially the same amino acid sequence asshown in FIG. 11. In another embodiment, the GALR2 receptor has theamino acid sequence shown in FIG. 11.

[0076] This invention provides a vector comprising the above-describednucleic acid molecule.

[0077] Vectors which comprise the isolated nucleic acid moleculedescribed hereinabove also are provided. Suitable vectors comprise, butare not limited to, a plasmid or a virus. These vectors may betransformed into a suitable host cell to form a host cell expressionsystem for the production of a polypeptide having the biologicalactivity of a galanin GALR2 receptor. Suitable host cells include, forexample, neuronal cells such as the glial cell line C6, a Xenopus cellsuch as an oocyte or melanophore cell, as well as numerous mammaliancells and non-neuronal cells.

[0078] This invention provides the above-described vector adapted forexpression in a bacterial cell which further comprises the regulatoryelements necessary for expression of the nucleic acid in the bacterialcell operatively linked to the nucleic acid encoding the GALR2 receptoras to permit expression thereof.

[0079] This invention provides the above-described vector adapted forexpression in a yeast cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the yeast celloperatively linked to the nucleic acid encoding the GALR2 receptor as topermit expression thereof.

[0080] This invention provides the above-described vector adapted forexpression in an insect cell which comprises the regulatory elementsnecessary for expression of the nucleic acid in the insect celloperatively linked to the nucleic acid encoding the GALR2 receptor as topermit expression thereof. In a still further embodiment, the vector isa baculovirus.

[0081] In one embodiment, the vector is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the mammalian GALR2 receptor as to permitexpression thereof.

[0082] In a further embodiment, the vector is adapted for expression ina mammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the rat GALR2 receptor as to permitexpression thereof.

[0083] In a still further embodiment, the vector is a plasmid.

[0084] In another embodiment, the plasmid is adapted for expression in amammalian cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the mammalian cell operatively linkedto the nucleic acid encoding the human GALR2 receptor as to permitexpression thereof.

[0085] This invention provides the above-described plasmid adapted forexpression in a mammalian cell which comprises the regulatory elementsnecessary for expression of nucleic acid in a mammalian cell operativelylinked to the nucleic acid encoding the mammalian GALR2 receptor as topermit expression thereof.

[0086] This invention provides a plasmid designated K985 (ATCC AccessionNo. 97426) which comprises the regulatory elements necessary forexpression of DNA in a mammalian cell operatively linked to DNA encodingthe GALR2 galanin receptor so as to permit expression thereof.

[0087] This plasmid (K985) was deposited on Jan. 24, 1996, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.97426.

[0088] This invention provides a plasmid designated BO29 (ATCC AccessionNo. 97735) which comprises the regulatory elements necessary forexpression of DNA in a mammalian cell operatively linked to DNA encodingthe GALR2 galanin receptor as to permit expression thereof.

[0089] This plasmid (BO29) was deposited on Sep. 25, 1996, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.97735.

[0090] This invention provides a plasmid designated K1045 (ATCCAccession No. 97778) which comprises the regulatory elements necessaryfor expression of DNA in a mammalian cell operatively linked to DNAencoding the GALR2 galanin receptor so as to permit expression thereof.

[0091] This plasmid (K1045) was deposited on Oct. 30, 1996, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.97426.

[0092] This invention provides a plasmid designated BO39 (ATCC AccessionNo. ______) which comprises the regulatory elements necessary forexpression of DNA in a mammalian cell operatively linked to DNA encodingthe GALR2 galanin receptor as to permit expression thereof.

[0093] This plasmid (BO39) was deposited on Jan. 15, 1997, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.______.

[0094] This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the receptor dependingupon the host cell used. In an embodiment, the vector or plasmidcomprises the coding sequence of the GALR2 receptor and the regulatoryelements necessary for expression in the host cell.

[0095] This invention provides a eukaryotic cell comprising theabove-described plasmid or vector. This invention provides a mammaliancell comprising the above-described plasmid or vector. In an embodimentthe cell is a Xenopus oocyte or melanophore cell. In an embodiment, thecell is a neuronal cell such as the glial cell line C6. In anembodiment, the mammalian cell is non-neuronal in origin. In anembodiment, the mammalian cell is a COS-7 cell. In another embodimentthe mammalian cell is a Chinese hamster ovary (CHO) cell. In anotherembodiment, the cell is a mouse Y1 cell.

[0096] In still another embodiment, the mammalian cell is a 293 humanembryonic kidney cell. In still another embodiment, the mammalian cellis a NIH-3T3 cell. In another embodiment, the mammalian cell is anLM(tk−) cell. In still another embodiment, the mammalian cell is theLM(tk−) cell designated L-rGALR2-8. This cell line was deposited withthe ATCC on Mar. 28, 1996, under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure, and was accorded ATCC Accession No.CRL-12074. In yet another embodiment, the mammalian cell is the LM(tk−)cell designated L-rGALR2I-4 (which comprises the intronless plasmidK1045). This cell line was deposited with the ATCC on Oct. 30, 1996,under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure, and was accorded ATCC Accession No. CRL-12223.

[0097] In another embodiment, the mammalian cell is the Chinese hamsterovary (CHO) cell designated C-rGalR2-79. C-rGalR2-79 expresses the ratGALR2 receptor and comprises a plasmid containing the intron within thecoding region. This cell line was deposited with the ATCC on Jan. 15,1997, under the provisions of the Budapest Treaty for the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure, and was accorded ATCC Accession No. CRL-12262.

[0098] This invention also provides an insect cell comprising theabove-described vector. In an embodiment, the insect cell is an Sf9cell. In another embodiment, the insect cell is an Sf21 cell.

[0099] This invention provides a membrane preparation isolated from anyof the above-described cells.

[0100] This invention provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid K985.

[0101] This invention further provides a nucleic acid probe comprisingat least 15 nucleotides, which probe specifically hybridizes with anucleic acid encoding a GALR2 receptor, wherein the probe has a uniquesequence corresponding to a sequence present within one of the twostrands of the nucleic acid encoding the GALR2 receptor contained inplasmid K1045.

[0102] This invention still further provides a nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a GALR2 receptor, wherein the probe has aunique sequence corresponding to a sequence present within (a) thenucleic acid sequence described in FIG. 1 or (b) the reverse complementthereto. This invention also provides a nucleic acid probe comprising atleast 15 nucleotides, which probe specifically hybridizes with a nucleicacid encoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid BO29. Thisinvention also provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid BO39.

[0103] This invention provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 10 (Seq. ID No. 29) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 10.

[0104] This invention provides a nucleic acid probe comprising at least15 nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within (a) the nucleic acid sequenceshown in FIG. 1 (Seq. I.D. No. 7) or (b) the reverse complement to thenucleic acid sequence shown in FIG. 1 (Seq. I.D. No. 7). In oneembodiment, the nucleic acid encoding a GALR2 receptor comprises anintron, the sequence of which intron is described in FIG. 3 (Seq. I.D.No. 9). In another embodiment, the nucleic acid encoding a GALR2receptor comprises an intron, the sequence of which intron is describedin FIG. 12C (Seq. I.D. No. 31).

[0105] This invention further provides a nucleic acid probe comprising anucleic acid molecule of at least 15 nucleotides which is complementaryto a unique fragment of the sequence of a nucleic acid molecule encodinga GALR2 receptor. This invention also provides a nucleic acid probecomprising a nucleic acid molecule of at least 15 nucleotides which iscomplementary to the antisense sequence of a unique fragment of thesequence of a nucleic acid molecule encoding a GALR2 receptor.

[0106] In one embodiment, the nucleic acid probe is DNA. In anotherembodiment the nucleic acid probe is RNA. As used herein, the phrase“specifically hybridizing” means the ability of a nucleic acid moleculeto recognize a nucleic acid sequence complementary to its own and toform double-helical segments through hydrogen bonding betweencomplementary base pairs.

[0107] This nucleic acid of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of a nucleic acid encoding theGALR2 galanin receptors can be used as a probe. Nucleic acid probetechnology is well known to those skilled in the art who will readilyappreciate that such probes may vary greatly in length and may belabeled with a detectable label, such as a radioisotope or fluorescentdye, to facilitate detection of the probe. DNA probe molecules may beproduced by insertion of a DNA molecule which encodes the GALR2 receptorinto suitable vectors, such as plasmids or bacteriophages, followed bytransforming into suitable bacterial host cells, replication in thetransformed bacterial host cells and harvesting of the DNA probes, usingmethods well known in the art. Alternatively, probes may be generatedchemically from DNA synthesizers.

[0108] RNA probes may be generated by inserting the DNA molecule whichencodes the GALR2 galanin receptor downstream of a bacteriophagepromoter such as T3, T7 or SP6. Large amounts of RNA probe may beproduced by incubating the labeled nucleotides with the linearizedfragment where it contains an upstream promoter in the presence of theappropriate RNA polymerase.

[0109] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to mRNA encoding a GALR2galanin receptor, so as to prevent translation of the mRNA.

[0110] This invention provides an antisense oligonucleotide having asequence capable of specifically hybridizing to the genomic DNA moleculeencoding a GALR2 receptor.

[0111] This invention provides an antisense oligonucleotide comprisingchemical analogues of nucleotides.

[0112] This invention provides an antibody directed to a GALR2 receptor.This invention also provides an antibody directed to a rat GALR2receptor. This invention also provides an antibody directed to a humanGALR2 receptor. In an embodiment, the human GALR2 has an amino acidsequence the same or substantially the same as an amino acid sequenceencoded by plasmid K985 or an amino acid sequence encoded by plasmidBO29. In another embodiment, the human GALR2 has an amino acid sequencethe same or substantially the same as an amino acid sequence encoded byplasmid BO39.

[0113] This invention provides a monoclonal antibody directed to anepitope of a GALR2 receptor, which epitope is present on the surface ofa cell expressing a GALR2 receptor.

[0114] This invention provides a pharmaceutical composition comprisingan amount of the oligonucleotide effective to reduce activity of a GALR2receptor by passing through a cell membrane and binding specificallywith mRNA encoding a GALR2 receptor in the cell so as to prevent itstranslation and a pharmaceutically acceptable carrier capable of passingthrough a cell membrane. In one embodiment, the oligonucleotide iscoupled to a substance which inactivates mRNA. In another embodiment,the substance which inactivates mRNA is a ribozyme.

[0115] This invention provides the above-described pharmaceuticalcomposition, wherein the pharmaceutically acceptable carrier capable ofpassing through a cell membrane comprises a structure which binds to areceptor specific for a selected cell type and is thereby taken up bycells of the selected cell type.

[0116] This invention provides a pharmaceutical composition comprisingan amount of an antagonist effective to reduce the activity of a GALR2receptor and a pharmaceutically acceptable carrier.

[0117] This invention provides a pharmaceutical composition comprisingan amount of an agonist effective to increase activity of a GALR2receptor and a pharmaceutically acceptable carrier.

[0118] This invention provides the above-described pharmaceuticalcomposition which comprises an amount of the antibody effective to blockbinding of a ligand to the GALR2 receptor and a pharmaceuticallyacceptable carrier.

[0119] As used herein, “pharmaceutically acceptable carriers” means anyof the standard pharmaceutically acceptable carriers. Examples include,but are not limited to, phosphate buffered saline, physiological saline,water and emulsions, such as oil/water emulsions.

[0120] This invention provides a transgenic nonhuman mammal expressingDNA encoding a GALR2 receptor.

[0121] This invention provides a transgenic nonhuman mammal comprising ahomologous recombination knockout of the native GALR2 receptor.

[0122] This invention provides a transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a GALR2 receptorso placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding a GALR2 receptor and which hybridizes tomRNA encoding a GALR2 receptor thereby reducing its translation.

[0123] This invention provides the above-described transgenic nonhumanmammal, wherein the DNA encoding a GALR2 receptor additionally comprisesan inducible promoter.

[0124] This invention provides the transgenic nonhuman mammal, whereinthe DNA encoding a GALR2 receptor additionally comprises tissue specificregulatory elements.

[0125] In an embodiment, the transgenic nonhuman mammal is a mouse.

[0126] Animal model systems which elucidate the physiological andbehavioral roles of GALR2 receptor are produced by creating transgenicanimals in which the activity of the GALR2 receptor is either increasedor decreased, or the amino acid sequence of the expressed GALR2 receptoris altered, by a variety of techniques. Examples of these techniquesinclude, but are not limited to: 1) Insertion of normal or mutantversions of DNA encoding a GALR2 receptor, by microinjection,electroporation, retroviral transfection or other means well known tothose skilled in the art, into appropriate fertilized embryos in orderto produce a transgenic animal or 2) Homologous recombination of mutantor normal, human or animal versions of these genes with the native genelocus in transgenic animals to alter the regulation of expression or thestructure of these GALR2 receptor sequences. The technique of homologousrecombination is well known in the art. It replaces the native gene withthe inserted gene and so is useful for producing an animal that cannotexpress native GALR2 receptors but does express, for example, aninserted mutant GALR2 receptor, which has replaced the native GALR2receptor in the animal's genome by recombination, resulting inunderexpression of the transporter. Microinjection adds genes to thegenome, but does not remove them, and so is useful for producing ananimal which expresses its own and added GALR2 receptors, resulting inoverexpression of the GALR2 receptors.

[0127] One means available for producing a transgenic animal, with amouse as an example, is as follows: Female mice are mated, and theresulting fertilized eggs are dissected out of their oviducts. The eggsare stored in an appropriate medium such as M2 medium. DNA or cDNAencoding a GALR2 receptor is purified from a vector by methods wellknown in the art. Inducible promoters may be fused with the codingregion of the DNA to provide an experimental means to regulateexpression of the trans-gene. Alternatively, or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipet puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only for exemplary purposes.

[0128] This invention provides a process for identifying a chemicalcompound which specifically binds to a GALR2 receptor which comprisescontacting cells containing DNA encoding and expressing on their cellsurface the GALR2 receptor, wherein such cells do not normally expressthe GALR2 receptor, with the compound under conditions suitable forbinding, and detecting specific binding of the chemical compound to theGALR2 receptor.

[0129] This invention further provides a process for identifying achemical compound which specifically binds to a GALR2 receptor whichcomprises contacting a membrane fraction from a cell extract of cellscontaining DNA encoding and expressing on their cell surface the GALR2receptor, wherein such cells do not normally express the GALR2 receptor,with the compound under conditions suitable for binding, and detectingspecific binding of the chemical compound to the GALR2 receptor.

[0130] This invention also provides a method for determining whether achemical compound can specifically bind to a GALR2 receptor whichcomprises contacting cells transfected with and expressing DNA encodingthe GALR2 receptor with the compound under conditions permitting bindingof compounds to such receptor, and detecting the presence of any suchcompound specifically bound to the GALR2 receptor, so as to therebydetermine whether the ligand specifically binds to the GALR2 receptor.

[0131] This invention provides a method for determining whether achemical compound can specifically bind to a GALR2 receptor whichcomprises preparing a cell extract from cells transfected with andexpressing DNA encoding the GALR2 receptor, isolating a membranefraction from the cell extract, contacting the membrane fraction withthe compound under conditions permitting binding of compounds to suchreceptor, and detecting the presence of the compound specifically boundto the GALR2 receptor, so as to thereby determine whether the compoundspecifically binds to the GALR2 receptor.

[0132] In one embodiment, the GALR2 receptor is a mammalian GALR2receptor. In another embodiment, the GALR2 receptor is a rat GALR2receptor. In still another embodiment, the GALR2 receptor has the sameor substantially the same amino acid sequence as that encoded by plasmidK985, or plasmid K1045. In still another embodiment, the GALR2 receptorhas the same or substantially the same amino acid sequence as the aminoacid sequence shown in FIG. 2 (Seq. I.D. No.8). In yet anotherembodiment, the GALR2 receptor has the amino acid sequence shown in FIG.2 (Seq. I.D. No. 8).

[0133] In another embodiment, the GALR2 receptor is a human GALR2receptor. In still another embodiment, the human GALR2 receptor has thesame or substantially the same amino acid sequence as the amino acidsequence encoded by plasmid BO29 or plasmid BO39. In yet anotherembodiment, the GALR2 receptor has the same or substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 11 (Seq.I.D. No. 30). In another embodiment, the GALR2 receptor has the aminoacid sequence shown in FIG. 11 (Seq. I.D. No. 30).

[0134] In one embodiment, the above process further comprisesdetermining whether the compound selectively binds to the GALR2 receptorrelative to another galanin receptor. In another embodiment, thedetermination whether the compound selectively binds to the GALR2receptor comprises: (a) determining the binding affinity of the compoundfor the GALR2 receptor and for such other galanin receptor; and (b)comparing the binding affinities so determined, the presence of a higherbinding affinity for the GALR2 receptor than for such other galaninreceptor inicating that the compound selectively binds to the GALR2receptor. In an embodiment, the other galanin receptor is a GALR1receptor. In another embodiment, the other galanin receptor is a GALR3receptor.

[0135] This invention provides a process for determining whether achemical compound is a GALR2 receptor agonist which comprises contactingcells transfected with and expressing DNA encoding the GALR2 receptorwith the compound under conditions permitting the activation of theGALR2 receptor, and detecting an increase in GALR2 receptor activity, soas to thereby determine whether the compound is a GALR2 receptoragonist.

[0136] This invention provides a process for determining whether achemical compound is a GALR2 receptor agonist which comprises preparinga cell extract from cells transfected with and expressing DNA encodingthe GALR2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the compound under conditionspermitting the activation of the GALR2 receptor, and detecting anincrease in GALR2 receptor activity, so as to thereby determine whetherthe compound is a GALR2 receptor agonist.

[0137] In one embodiment, the GALR2 receptor is a mammalian GALR2receptor. In another embodiment, the GALR2 receptor is a rat GALR2receptor. In still another embodiment, the GALR2 receptor has the sameor substantially the same amino acid sequence as that encoded by plasmidK985, or plasmid K1045. In still another embodiment, the GALR2 receptorhas the same or substantially the same amino acid sequence as the aminoacid sequence shown in FIG. 2 (Seq. I.D. No.8). In yet anotherembodiment, the GALR2 receptor has the amino acid sequence shown in FIG.2 (Seq. I.D. No. 8).

[0138] In another embodiment, the GALR2 receptor is a human GALR2receptor. In still another embodiment, the human GALR2 receptor has thesame or substantially the same amino acid sequence as the amino acidsequence encoded by plasmid BO29 or plasmid BO39. In yet anotherembodiment, the GALR2 receptor has the same or substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 11 (Seq.I.D. No. 30). In another embodiment, the GALR2 receptor has the aminoacid sequence shown in FIG. 11 (Seq. I.D. No. 30).

[0139] This invention provides a process for determining whether achemical compound is a GALR2 receptor antagonist which comprisescontacting cells transfected with and expressing DNA encoding the GALR2receptor with the compound in the presence of a known GALR2 receptoragonist, such as galanin, under conditions permitting the activation ofthe GALR2 receptor, and detecting a decrease in GALR2 receptor activity,so as to thereby determine whether the compound is a GALR2 receptorantagonist.

[0140] This invention provides a process for determining whether achemical compound is a GALR2 receptor antagonist which comprisespreparing a cell extract from cells transfected with and expressing DNAencoding the GALR2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the ligand in thepresence of a known GALR2 receptor agonist, such as galanin, underconditions permitting the activation of the GALR2 receptor, anddetecting a decrease in GALR2 receptor activity, so as to therebydetermine whether the compound is a GALR2 receptor antagonist.

[0141] In one embodiment, the GALR2 receptor is a mammalian GALR2receptor. In another embodiment, the GALR2 receptor is a rat GALR2receptor. In still another embodiment, the GALR2 receptor has the sameor substantially the same amino acid sequence as that encoded by plasmidK985, or plasmid K1045. In still another embodiment, the GALR2 receptorhas the same or substantially the same amino acid sequence as the aminoacid sequence shown in FIG. 2 (Seq. I.D. No.8). In yet anotherembodiment, the GALR2 receptor has the amino acid sequence shown in FIG.2 (Seq. I.D. No. 8).

[0142] In another embodiment, the GALR2 receptor is a human GALR2receptor. In still another embodiment, the human GALR2 receptor has thesame or substantially the same amino acid sequence as the amino acidsequence encoded by plasmid BO29 or plasmid BO39. In yet anotherembodiment, the GALR2 receptor has the same or substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 11 (Seq.I.D. No. 30). In another embodiment, the GALR2 receptor has the aminoacid sequence shown in FIG. 11 (Seq. I.D. No. 30).

[0143] In an embodiment of the above-described methods, the cell is aninsect cell. In another embodiment, the cell is a mammalian cell. In afurther embodiment, the cell is non-neuronal in origin. In still furtherembodiments, the non-neuronal cell is a COS-7 cell, 293 human embryonickidney cell, NIH-3T3 cell, a CHO cell, or LM(tk−) cell. In yet anotherembodiment of any of the processes of this invention the cell is theLM(tk−) cell L-rGALR2-8 (ATCC Accession No. CRL-12074), the LM(tk−) cellL-rGALR2I-4 (ATCC Accession No. CRL-12223, or the CHO cell C-rGalR2-79(ATCC Accession No. CRL-12262).

[0144] This invention provides a compound determined by theabove-described processes. In one embodiment of the above-describedprocesses, the compound is not previously known to bind to a GALR2receptor.

[0145] This invention provides a GALR2 agonist determined by theabove-described processes. This invention also provides a GALR2antagonist determined by the above-described processes.

[0146] This invention provides a pharmaceutical composition whichcomprises an amount of a GALR2 receptor agonist effective to increaseactivity of a GALR2 receptor and a pharmaceutically acceptable carrier.

[0147] This invention provides a pharmaceutical composition whichcomprises an amount of a GALR2 receptor antagonist effective to reduceactivity of a GALR2 receptor and a pharmaceutically acceptable carrier.

[0148] In further embodiments of the above-described processes, theagonist or antagonist is not previously known to bind to a GALR2receptor.

[0149] This invention provides a process involving competitive bindingfor identifying a chemical compound which specifically binds to a GALR2receptor, which comprises separately contacting cells expressing ontheir cell surface the GALR2 receptor, wherein such cells do notnormally express the GALR2 receptor, with both the chemical compound anda second chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the GALR2 receptor, a decrease in the binding of the second chemicalcompound to the GALR2 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the GALR2 receptor.

[0150] This invention further provides a process involving competitivebinding for identifying a chemical compound which specifically binds toa human GALR2 receptor, which comprises separately contacting a membranefraction from a cell extract of cells expressing on their cell surfacethe GALR2 receptor, wherein such cells do not normally express the GALR2receptor, with both the chemical compound and a second chemical compoundknows to bind to the receptor, and with only the second chemicalcompound, under conditions suitable for binding of both compounds, anddetecting specific binding of the chemical compound to the GALR2receptor, a decrease in the binding of the second chemical compound tothe GALR2 receptor in the presence of the chemical compound indicatingthat the chemical compound binds to the GALR2 receptor.

[0151] This invention further provides a process for determining whethera chemical compound specifically binds to and activates a GALR2receptor, which comprises contacting cells producing a second messengerresponse and expressing on their cell surface the GALR2 receptor,wherein such cells do not normally express the GALR2 receptor, with thechemical compound under conditions suitable for activation of the GALR2receptor, and measuring the second messenger response in the presenceand in the absence of the chemical compound, a change in the secondmessenger response in the presence of the chemical compound indicatingthat the compound activates the GALR2 receptor.

[0152] This invention further provides a process for determining whethera chemical compound specifically binds to and activates a GALR2receptor, which comprises contacting a membrane fraction from a cellextract of cells producing a second messenger response and expressing ontheir cell surface the GALR2 receptor, wherein such cells do notnormally express the GALR2 receptor, with the chemical compound underconditions suitable for activation of the GALR2 receptor, and measuringthe second messenger response in the presence and in the absence of thechemical compound, a change in the second messenger response in thepresence of the chemical compound indicating that the compound activatesthe GALR2 receptor.

[0153] In one embodiment of the above processes, the second messengerresponse comprises adenylate cyclase activity and the change in secondmessenger response is a decrease in adenylate cyclase activity. In oneembodiment, adenylate cyclase activity is determined by measurement ofcyclic AMP levels.

[0154] In another embodiment of the above processes, the secondmessenger response comprises arachidonic acid release and the change insecond messenger response is an increase in arachidonic acid levels.

[0155] In another embodiment of the above processes, the secondmessenger response comprises intracellular calcium levels and the changein second messenger response is an increase in intracellular calciumlevels.

[0156] In a still further embodiment of the above processes, the secondmessenger response comprises inositol phospholipid hydrolysis and thechange in second messenger response is an increase in inositolphospholipid hydrolysis.

[0157] This invention further provides a process for determining whethera chemical compound specifically binds to and inhibits activation of aGALR2 receptor, which comprises separately contacting cells producing asecond messenger response and expressing on their cell surface the GALR2receptor, wherein such cells do not normally express the GALR2 receptor,with both the chemical compound and a second chemical compound known toactivate the GALR2 receptor, and with only the second compound, underconditions suitable for activation of the GALR2 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR2 receptor.

[0158] This invention further provides a process for determining whethera chemical compound specifically binds to and inhibits activation of aGALR2 receptor, which comprises separately contacting a membranefraction from a cell extract of cells producing a second messengerresponse and expressing on their cell surface the GALR2 receptor,wherein such cells do not normally express the GALR2 receptor, with boththe chemical compound and a second chemical compound known to activatethe GALR2 receptor, and with only the second chemical compound, underconditions suitable for activation of the GALR2 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR2 receptor.

[0159] In one embodiment of the above processes, the second messengerresponse comprises adenylate cyclase activity and the change in secondmessenger response is a smaller decrease in the level of adenylatecyclase activity in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound. In one embodiment, adenylate cyclase activity isdetermined by measurement of cyclic AMP levels.

[0160] In another embodiment of the above processes the second messengerresponse comprises arachidonic acid release, and the change in secondmessenger response is a smaller increase in arachidonic acid levels inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compound.

[0161] In another embodiment of the above processes the second messengerresponse comprises intracellular calcium levels, and the change insecond messenger response is a smaller increase in intracellular calciumlevels in the presence of both the chemical compound and the secondchemical compound than in the presence of only the second chemicalcompound.

[0162] In yet another embodiment of the above processes, the secondmessenger response comprises inositol phospholipid hydrolysis, and thechange in second messenger response is a smaller increase in inositolphospholipid hydrolysis in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound.

[0163] In an embodiment of any of the above processes, the GALR2receptor is a mammalian GALR2 receptor. In another embodiment of theabove processes, the GALR2 receptor is a rat GALR2 receptor or a humanGALR2 receptor. In still another embodiment of the above processes, theGALR2 receptor has the same or substantially the same amino acidsequence as encoded by the plasmid K985 ATCC Accession No. 97426), orplasmid K1045 (ATCC Accession No. 97778). In a still further embodimentof the above processes, the GALR2 receptor has the same or substantiallythe same amino acid sequence as that shown in FIG. 2 (Seq. ID No. 8). Inanother embodiment of the above processes, the GALR2 receptor has thesame or substantially the same amino acid sequence as the amino acidsequence encoded by the plasmid BO29 (ATCC Accession No. 97735) or theplasmid BO39 (ATCC Accession No. ______). In a still further embodimentof the above processes, the GALR2 receptor has the same or substantiallythe same amino acid sequence as that shown in FIG. 11 (Seq. ID No. 30).

[0164] In an embodiment of any of the above processes, the cell is aninsect cell. In another embodiment of any of the above processes, thecell is a mammalian cell. In still further embodiments, the cell isnonneuronal in origin.

[0165] In another embodiment of the above processes, the nonneuronalcell is a COS-7 cell, 293 human embryonic kidney cell, NIH-3T3 cell, amouse Y1 cell or LM(tk−) cell. In still further embodiments, nonneuronalcell is the LM(tk−) cell designated L-rGALR2-8 (ATCC Accession No.CRL-12074), the LM(tk−) cell L-rGALR2I-4 (ATCC Accession No. CRL-12223,or the CHO cell C-rGalR2-79 (ATCC Accession No. ______).

[0166] This invention further provides a compound determined by any ofthe above processes. In another embodiment, the compound is notpreviously known to bind to a GALR2 receptor.

[0167] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a GALR2 receptor to identify acompound which specifically binds to the GALR2 receptor, which comprises(a) contacting cells transfected with and expressing DNA encoding theGALR2 receptor with a compound known to bind specifically to the GALR2receptor; (b) contacting the preparation of step (a) with the pluralityof compounds not known to bind specifically to the GALR2 receptor, underconditions permitting binding of compounds known to bind the GALR2receptor; (c) determining whether the binding of the compound known tobind to the GALR2 receptor is reduced in the presence of the compounds,relative to the binding of the compound in the absence of the pluralityof compounds; and if so (d) separately determining the binding to theGALR2 receptor of each compound included in the plurality of compounds,so as to thereby identify the compound which specifically binds to theGALR2 receptor.

[0168] This invention provides a method of screening a plurality ofchemical compounds not known to bind to a GALR2 receptor to identify acompound which specifically binds to the GALR2 receptor, which comprises(a) preparing a cell extract from cells transfected with and expressingDNA encoding the GALR2 receptor, isolating a membrane fraction from thecell extract, contacting the membrane fraction with a compound known tobind specifically to the GALR2 receptor; (b) contacting the preparationof step (a) with the plurality of compounds not known to bindspecifically to the GALR2 receptor, under conditions permitting bindingof compounds known to bind the GALR2 receptor; (c) determining whetherthe binding of the compound known to bind to the GALR2 receptor isreduced in the presence of the compounds, relative to the binding of thecompound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the GALR2 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the GALR2 receptor.

[0169] In one embodiment of the above-described methods, the GALR2receptor is a rat GALR2 receptor. In another embodiment, the GALR2receptor has the same or substantially the same amino acid sequence asthe amino acid sequence shown in FIG. 2 (Seq. I.D. No. 8). In yetanother embodiment, the GALR2 receptor has the amino acid sequence shownin FIG. 2 (Seq. I.D. No. 8). In another embodiment, the GALR2 receptoris a human GALR2 receptor. In still another embodiment, the GALR2receptor has the same or substantially the same amino acid sequence asthe amino acid sequence encoded by plasmid BO29 or plasmid BO39. Inanother embodiment, the GALR2 receptor has the same or substantially thesame amino acid sequence as the amino acid sequence shown in FIG. 11(Seq. I.D. No. 30). In yet another embodiment, the GALR2 receptor hasthe amino acid sequence shown in FIG. 11 (Seq. I.D. No. 30).

[0170] This invention provides a method of screening a plurality ofchemical compounds not known to activate a GALR2 receptor to identify acompound which activates the GALR2 receptor which comprises (a)contacting cells transfected with and expressing the GALR2 receptor withthe plurality of compounds not known to activate the GALR2 receptor,under conditions permitting activation of the GALR2 receptor; (b)determining whether the activity of the GALR2 receptor is increased inthe presence of the compounds; and if so (c) separately determiningwhether the activation of the GALR2 receptor is increased by eachcompound included in the plurality of compounds, so as to therebyidentify the compound which activates the GALR2 receptor.

[0171] This invention provides a method of screening a plurality ofchemical compounds not known to activate a GALR2 receptor to identify acompound which activates the GALR2 receptor which comprises (a)preparing a cell extract from cells transfected with and expressing DNAencoding the GALR2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the plurality ofcompounds not known to activate the GALR2 receptor, under conditionspermitting activation of the GALR2 receptor; (b) determining whether theactivity of the GALR2 receptor is increased in the presence of thecompounds; and if so (c) separately determining whether the activationof the GALR2 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the GALR2 receptor.

[0172] In an embodiment of the above-described methods, the GALR2receptor is a rat GALR2 receptor. In still another embodiment, the GALR2receptor has the same or substantially the same amino acid sequence asthe amino acid sequence shown in FIG. 2 (Seq. I.D. No.8). In yet anotherembodiment, the GALR2 receptor has the amino acid sequence shown in FIG.2 (Seq. I.D. No. 8). In another embodiment, the GALR2 receptor is ahuman GALR2 receptor. In still another embodiment, the GALR2 receptorhas the same or substantially the same amino acid sequence as the aminoacid sequence encoded by plasmid BO29 or plasmid BO39. In anotherembodiment, the GALR2 receptor has the same or substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 11 (Seq.I.D. No. 30). In yet another embodiment, the GALR2 receptor has theamino acid sequence shown in FIG. 11 (Seq. I.D. No. 30).

[0173] This invention provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a GALR2receptor to identify a compound which inhibits the activation of theGALR2 receptor, which comprises (a) contacting cells transfected withand expressing the GALR2 receptor with the plurality of compounds in thepresence of a known GALR2 receptor agonist, under conditions permittingactivation of the GALR2 receptor; (b) determining whether the activationof the GALR2 receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the GALR2 receptor in theabsence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the GALR2 receptor for eachcompound included in the plurality of compounds, so as to therebyidentify the compound which inhibits the activation of the GALR2receptor.

[0174] This invention provides a method of screening a plurality ofchemical compounds not known to inhibit the activation of a GALR2receptor to identify a compound which inhibits the activation of theGALR2 receptor, which comprises (a) preparing a cell extract from cellstransfected with and expressing DNA encoding the GALR2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with the plurality of compounds in the presence of aknown GALR2 receptor agonist, under conditions permitting activation ofthe GALR2 receptor; (b) determining whether the activation of the GALR2receptor is reduced in the presence of the plurality of compounds,relative to the activation of the GALR2 receptor in the absence of theplurality of compounds; and if so (c) separately determining theinhibition of activation of the GALR2 receptor for each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which inhibits the activation of the GALR2 receptor.

[0175] In an embodiment of the above-described methods, the GALR2receptor is a rat GALR2 receptor. In another embodiment, the GALR2receptor has the same or substantially the same amino acid sequence asthe amino acid sequence shown in FIG. 2 (Seq. I.D. No.8). In yet anotherembodiment, the GALR2 receptor has the amino acid sequence shown in FIG.2 (Seq. I.D. No. 8). In another embodiment, the GALR2 receptor is ahuman GALR2 receptor. In still another embodiment, the GALR2 receptorhas the same or substantially the same amino acid sequence as the aminoacid sequence encoded by plasmid BO29 or plasmid BO39. In anotherembodiment, the GALR2 receptor has the same or substantially the sameamino acid sequence as the amino acid sequence shown in FIG. 11 (Seq.I.D. No. 30). In yet another embodiment, the GALR2 receptor has theamino acid sequence shown in FIG. 11 (Seq. I.D. No. 30).

[0176] In one embodiment of any of the above-described methods, theactivation of the GALR2 receptor is determined by a second messengerassay. In an embodiment, the second messenger assay measures adenylatecyclase activity. In other embodiments, the second messenger is cyclicAMP, intracellular calcium, or arachidonic acid or a phosphoinositollipid metabolite. Second messenger coupling may also be measured byassaying the binding of GTP gamma S to membranes.

[0177] This invention further provides a method of measuring GALR2receptor activation in an oocyte expression system such as a Xenopusoocyte or melanophore. In an embodiment, receptor activation isdetermined by measurement of ion channel activity.

[0178] Expression of genes in Xenopus oocytes is well known in the art(A. Coleman, Transcription and Translation: A Practical Approach (B. D.Hanes, S. J. Higgins, eds., pp 271-302, IRL Press, Oxford, 1984; Y. Masuet al., Nature 329:21583-21586, 1994) and is performed usingmicroinjection of native mRNA or in vitro synthesized mRNA into frogoocytes. The preparation of in vitro synthesized mRNA can be performedby various standard techniques (J. Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1989) including using T7 polymerase with the mCAPRNA capping kit (Stratagene).

[0179] In a further embodiment of the invention, the cell is a mammaliancell. In another embodiment of the invention, the mammalian cell isnon-neuronal in origin. In still further embodiments of the invention,the non-neuronal cell is a COS-7 cell, a 293 human embryonic kidneycell, a mouse Y1 cell, a LM(tk−) cell, a CHO cell, or an NIH-3T3 cell.In an embodiment of the invention, the nonneuronal cell is the LM(tk−)cell designated L-rGALR2-8 (ATCC Accession No. CRL-12074), the LM(tk−)cell L-rGALR2I-4 (ATCC Accession No. CRL-12223, or the CHO cellC-rGalR2-79 (ATCC Accession No. ______).

[0180] This invention provides a pharmaceutical composition comprising acompound identified by the above-described methods and apharmaceutically acceptable carrier.

[0181] In an embodiment of the above-described methods, the cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, 293 human embryonic kidney cell, NIH-3T3 cell, a mouseY1 cell or LM(tk−) cell.

[0182] In one embodiment of the above-described methods, the compound isnot previously known.

[0183] This invention provides a GALR2 receptor agonist detected by theabove-described methods. This invention provides a GALR2 receptorantagonist detected by the above-described methods. In an embodiment thecell is a non-mammalian cell, for example, a Xenopus oocyte ormelanophore. In another embodiment the cell is a neuronal cell, forexample, a glial cell line such as C6. In an embodiment, the cell isnon-neuronal in origin. In a further embodiment, the cell is a Cos-7 ora CHO cell, a 293 human embryonic kidney cell, an LM(tk−) cell or anNIH-3T3 cell. In an embodiment of the invention, the LM(tk−) cell is thecell designated L-rGALR2-8 (ATCC Accession No. CRL-12074), the LM(tk−)cell L-rGALR2I-4 (ATCC Accession No. CRL-12223, or the CHO cellC-rGalR2-79 (ATCC Accession No. ______).

[0184] This invention provides a pharmaceutical composition comprising adrug candidate identified by the above-described methods and apharmaceutically acceptable carrier.

[0185] This invention provides a method for determining whether achemical compound is a GALR2 antagonist which comprises: (a)administering to an animal a GALR2 agonist and measuring the amount offood intake in the animal; (b) administering to a second animal both theGALR2 agonist and the chemical compound, and measuring the amount offood intake in the second animal; and (c) determining whether the amountof food intake is reduced in the presence of the chemical compoundrelative to the amount of food intake in the absence of the compound, soas to thereby determine whether the compound is a GALR2 antagonist. Thisinvention further provides a method of screening a plurality of chemicalcompounds to identify a chemical compound which is a GALR2 antagonistwhich comprises: (a) administering to an animal a GALR2 agonist andmeasuring the amount of food intake in the animal; (b) administering toa second animal the GALR2 agonist and at least one chemical compound ofthe plurality of compounds, and measuring the amount of food intake inthe animal; (c) determining whether the amount of food intake is reducedin the presence of at least one chemical compound of the plurality ofchemical compounds relative to the amount of food intake in the absenceof at least one of the compounds, and if so; (d) separately determiningwhether each chemical compound is a GALR2 antagonist according to themethod described above, so as to thereby determine if the chemicalcompound is a GALR2 antagonist. In one embodiment the GALR2 agonist is[D-Trp]₂-galanin₍₁₋₂₉₎. In another embodiment the animal is a non-humanmammal. In a further embodiment, the animal is a rodent.

[0186] This invention provides a method of detecting expression of aGALR2 receptor by detecting the presence of mRNA coding for the GALR2receptor which comprises obtaining total mRNA from a cell or tissuesample and contacting the mRNA so obtained with the above-describednucleic acid probe under hybridizing conditions, detecting the presenceof mRNA hybridized to the probe, and thereby detecting the expression ofthe GALR2 receptor by the cell or in the tissue.

[0187] This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by administering to thesubject an amount of a GALR2 selective compound, effective to treat theabnormality. Abnormalities which may be treated include cognitivedisorder, pain, sensory disorder (olfactory, visual), motor coordinationabnormality, motion sickness, neuroendocrine disorders, sleep disorders,migraine, Parkinson's disease, hypertension, heart failure,convulsion/epilepsy, traumatic brain injury, diabetes, glaucoma,electrolyte imbalances, respiratory disorders (asthma, emphysema),depression, reproductive disorders, gastric and intestinal ulcers,gastroesophageal reflux disorder, gastric hypersecretion,gastrointestinal motility disorders (diarrhea), inflammation, immunedisorders, and anxiety. In one embodiment the compound is an agonist. Inanother embodiment the compound is an antagonist.

[0188] This invention provides a method of treating an abnormality in asubject, wherein the abnormality is alleviated by the inhibition of aGALR2 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition effective todecrease the activity of the GALR2 receptor in the subject, therebytreating the abnormality in the subject. In an embodiment, theabnormality is obesity. In another embodiment, the abnormality isbulimia.

[0189] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by the activation of aGALR2 receptor which comprises administering to a subject an effectiveamount of the above-described pharmaceutical composition effective toactivate the GALR2 receptor in the subject. In an embodiment, theabnormal condition is anorexia.

[0190] In another embodiment, the compound binds selectively to a GALR2receptor. In yet another embodiment, the compound binds to the GALR2receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to a GALR1 receptor. In a still furtherembodiment, the compound binds to the GALR2 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to a GALR3 receptor.

[0191] This invention provides a method of detecting the presence of aGALR2 receptor on the surface of a cell which comprises contacting thecell with the above-described antibody under conditions permittingbinding of the antibody to the receptor, detecting the presence of theantibody bound to the cell, and thereby detecting the presence of aGALR2 receptor on the surface of the cell.

[0192] This invention provides a method of determining the physiologicaleffects of varying levels of activity of GALR2 receptors which comprisesproducing a transgenic nonhuman mammal whose levels of GALR2 receptoractivity are varied by use of an inducible promoter which regulatesGALR2 receptor expression.

[0193] This invention provides a method of determining the physiologicaleffects of varying levels of activity of GALR2 receptors which comprisesproducing a panel of transgenic nonhuman mammals each expressing adifferent amount of GALR2 receptor.

[0194] This invention provides a method for identifying an antagonistcapable of alleviating an abnormality wherein the abnormality isalleviated by decreasing the activity of a GALR2 receptor comprisingadministering a compound to the above-described transgenic nonhumanmammal and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic nonhuman mammal asa result of overactivity of a GALR2 receptor, the alleviation of theabnormality identifying the compound as an antagonist.

[0195] This invention provides an antagonist identified by theabove-described methods. This invention provides a pharmaceuticalcomposition comprising an antagonist identified by the above-describedmethods and a pharmaceutically acceptable carrier.

[0196] This invention provides a method of treating an abnormality in asubject wherein the abnormality is alleviated by decreasing the activityof a GALR2 receptor which comprises administering to a subject aneffective amount of the above-described pharmaceutical composition,thereby treating the abnormality.

[0197] This invention provides a method for identifying an agonistcapable of alleviating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a GALR2 receptorcomprising administering a compound to a transgenic nonhuman mammal anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal, thealleviation of the abnormality identifying the compound as an agonist.

[0198] This invention provides an agonist identified by theabove-described methods.

[0199] This invention provides a pharmaceutical composition comprisingan agonist identified by the above-described methods and apharmaceutically acceptable carrier.

[0200] This invention provides a method for treating an abnormality in asubject wherein the abnormality is alleviated by increasing the activityof a GALR2 receptor which comprises administering to a subject aneffective amount of the above-described pharmaceutical composition,thereby treating the abnormality.

[0201] This invention provides a method for diagnosing a predispositionto a disorder associated with the activity of a specific human GALR2receptor allele which comprises: (a) obtaining DNA of subjects sufferingfrom the disorder; (b) performing a restriction digest of the DNA with apanel of restriction enzymes; (c) electrophoretically separating theresulting DNA fragments on a sizing gel; (d) contacting the resultinggel with a nucleic acid probe capable of specifically hybridizing with aunique sequence included within the sequence of a nucleic acid moleculeencoding a human GALR2 receptor and labelled with a detectable marker;(e) detecting labelled bands which have hybridized to DNA encoding ahuman GALR2 receptor labelled with a detectable marker to create aunique band pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps a-e; and (g)comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.

[0202] In an embodiment, a disorder associated with the activity of aspecific human GALR2 receptor allele is diagnosed. In anotherembodiment, the above-described method may be used to identify apopulation of patients having a specific GALR2 receptor allele, in whichpopulation the disorder may be alleviated by administering to thesubjects a GALR2-selective compound.

[0203] This invention provides a method of preparing the purified GALR2receptor which comprises: (a) inducing cells to express GALR2 receptor;(b) recovering the receptor from the induced cells; and (c) purifyingthe receptor so recovered.

[0204] This invention provides a method of preparing a purified GALR2receptor which comprises: (a) inserting nucleic acid encoding the GALR2receptor in a suitable vector; (b) introducing the resulting vector in asuitable host cell; (c) placing the resulting cell in suitable conditionpermitting the production of the isolated GALR2 receptor; (d) recoveringthe receptor produced by the resulting cell; and (e) purifying thereceptor so recovered.

[0205] This invention provides a method of modifying feeding behavior ofa subject which comprises administering to the subject an amount of acompound which is a galanin receptor agonist or antagonist effective toincrease or decrease the consumption of food by the subject so as tothereby modify feeding behavior of the subject. In one embodiment, thecompound is a GALR2 receptor antagonist and the amount is effective todecrease the consumption of food by the subject. In another embodimentthe compound is administered in combination with food.

[0206] In yet another embodiment the compound is a GALR2 receptoragonist and the amount is effective to increase the consumption of foodby the subject. In a still further embodiment, the compound isadministered in combination with food. In other embodiments the subjectis a vertebrate, a mammal, a human or a canine.

[0207] In one embodiment, the compound binds selectively to a GALR2receptor. In another embodiment, the compound binds to the GALR2receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to a GALR1 receptor. In anotherembodiment, the compound binds to the GALR2 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to a GALR3 receptor. In yet another embodiment, the compound bindsto the GALR2 receptor with an affinity greater than one hundred-foldhigher than the affinity with which the compound binds to a GALR1receptor. In another embodiment, the compound binds to the GALR2receptor with an affinity greater than one hundred-fold higher than theaffinity with which the compound binds to a GALR3 receptor.

[0208] This invention provides a method of treating Alzheimer's diseasein a subject which comprises administering to the subject an amount of acompound which is a galanin receptor antagonist effective to treat thesubject's Alzheimer's disease. In one embodiment, the galanin receptorantagonist is a GALR2 receptor antagonist and the amount of the compoundis effective to treat the subject's Alzheimer's disease.

[0209] This invention provides a method of producing analgesia in asubject which comprises administering to the subject an amount of acompound which is a galanin receptor agonist effective to produceanalgesia in the subject. In another embodiment, the galanin receptoragonist is a GALR2 receptor agonist and the amount of the compound iseffective to produce analgesia in the subject.

[0210] This invention provides a method of decreasing nociception in asubject which comprises administering to the subject an amount of acompound which is a GALR2 receptor agonist effective to decreasenociception in the subject.

[0211] This invention provides a method of treating pain in a subjectwhich comprises administering to the subject an amount of a compoundwhich is a GALR2 receptor agonist effective to treat pain in thesubject.

[0212] This invention provides a method of decreasing feeding behaviorof a subject which comprises administering a compound which is a GALR2receptor antagonist and a compound which is a Y5 receptor antagonist,the amount of such antagonists being effective to decrease the feedingbehavior of the subject. In one embodiment, the GALR2 antagonist and theY5 antagonist are administered in combination. In another embodiment,the GALR2 antagonist and the Y5 antagonist are administered once. Inanother embodiment, the GALR2 antagonist and the Y5 antagonist areadministered separately. In still another embodiment, the GALR2antagonist and the Y5 antagonist are administered once. In anotherembodiment, the galanin receptor antagonist is administered for about 1week to 2 weeks. In another embodiment, the Y5 receptor antagonist isadministered for about 1 week to 2 weeks.

[0213] In yet another embodiment, the GALR2 antagonist and the Y5antagonist are administered alternately. In another embodiment, theGALR2 antagonist and the Y5 antagonist are administered repeatedly. In astill further embodiment, the galanin receptor antagonist isadministered for about 1 week to 2 weeks. In another embodiment, the Y5receptor antagonist is administered for about 1 week to 2 weeks.

[0214] This invention also provides a method as described above, whereinthe compound is administered in a pharmaceutical composition comprisinga sustained release formulation.

[0215] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

[0216] Experimental Details

[0217] Materials and Methods

[0218] Construction and Screening of a Rat Hypothalamus cDNA Library

[0219] Total RNA was prepared from rat hypothalami by a modification ofthe guanidine thiocyanate method (Chirgwin, 1979). Poly A⁺ RNA waspurified using a FastTrack kit (Invitrogen Corp., San Diego, Calif.).Double stranded (ds) cDNA was synthesized from 4.6 μg of poly A⁺ RNAaccording to Gubler and Hoffman (1983) with minor modifications. Theresulting cDNA was ligated to BstXI/EcoRI adaptors (Invitrogen Corp.)and the excess adaptors removed by exclusion column chromatography. Highmolecular weight fractions of size-selected ds-cDNA were ligated inpEXJ.T7 (an Okayama and Berg expression vector modified from pcEXV(Miller & Germain, 1986) to contain BstXI and other additionalrestriction sites and a T7 promoter (Stratagene) and electroporated inE.coli MC 1061 (Gene Pulser, Biorad). A total of 3×10⁶ independentclones with a mean insert size of 2.2 kb were generated. The library wasplated on agar plates (Ampicillin selection) in 584 primary pools of˜5,000 independent clones. After 18 hours amplification, the bacteriafrom each pool were scraped, resuspended in 4 mL of LB media and 0.75 mLprocessed for plasmid purification (QIAwell-96 ultra, Qiagen,Inc.,Chatsworth, Calif.). Aliquots of each bacterial pool were stored at −85°C. in 20% glycerol.

[0220] To screen the library, COS-7 cells were plated in slide chambers(Lab-Tek) in Dulbecco's modified Eagle medium (DMEM) supplemented with10% calf serum, 100 U/mL of penicillin, 100 ug/mL streptomycin, 2 mML-glutamine (DMEM-C) and grown at 37° C. in a humidified 5% Co₂atmosphere for 24 hours before transfection. Cells were transfected withminiprep DNA prepared from the primary pools (˜4,500 cfu/pool) of therat hypothalamus cDNA library using a modification of the DEAE-dextranmethod (Warden & Thorne, 1968). Pools containing GALR1 were identifiedby PCR prior to screening and were omitted from the primary screen. Thegalanin binding assay was carried out after 48 hours. Cells were rinsedtwice with phosphate-buffered saline (PBS) then incubated with 1 nM¹²⁵I-porcine galanin (NEN; specific activity ˜2200 Ci/mmol) in 20 mMHEPES-NaOH, pH 7.4, containing 1.26 mM CaCl₂, 0.81 mM MgSO₄, 0.44 mMKH₂PO₄, 5.4 mM KCl, 10 mM NaCl, 0.1% BSA, and 0.1% bacitracin for onehour at room temperature. After rinsing and fixation in 2.5%glutaraldehyde, slides were rinsed in PBS, air-dried, and dipped inphotoemulsion (Kodak, NTB-2). After a 3-4 day exposure slides weredeveloped in Kodak D19 developer, fixed, and coverslipped (Aqua-Mount,Lerner Laboratories), then inspected for positive cells by brightfieldmicroscopy (Leitz Laborlux, 25× magnification). One pool with positivecells, (J126) was subdivided and rescreened repeatedly until a singlecolony was isolated that conferred galanin binding. The 3.8 kb cDNA ispreferably sequenced on both strands using Sequenase (US Biochemical,Cleveland, Ohio) according to the manufacturer. Nucleotide and peptidesequence analyses are performed using the Wisconsin Package (GCG,Genetics Computer group, Madison, Wis.) or PC/GENE (Intelligenetics,Mountain View, Calif.).

[0221] PCR Methodology

[0222] PCR reactions were carried out in 20 μl volumes using TaqPolymerase (Boehringer Mannheim, Indianapolis, Ind.) in a buffercontaining 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂ 0.01%gelatin, 0.2 mM each dNTP, and 1 μM each PCR primer. To prescreenlibrary pools for GALR1, two GALR1 primer sets were used (KS-1177/1178and KS-1311/1313, see below) to determine whether GALR1 was present inoriginal bacterial stocks of each library pool. PCR was carried out for40 cycles of 94° C./2 min, 68° C./2 min, 72° C./3 min. Pools positivefor GALR1 by PCR were eliminated from the library screen.

[0223] To confirm that the purified cDNA conferring galanin binding wasdistinct from GALR1, the isolated clone representing pool J126-10-334(K985) was subjected to PCR analysis using three GALR1 primer setsrepresenting different regions of GALR1. The nucleotide sequences of theprimer sets are shown below:

[0224] KS-1177: 5′-TGG GCA ACA GCC TAG TGA TCA CCG-3′ (Seq. I.D. No. 1)Nucleotides 146-169 of human GALR1 coding region, forward primer.

[0225] KS-1178: 5′-CTG CTC CCA GCA GAA GGT CTG GTT-3′ (Seq. I.D. No. 2)Nucleotides 547-570 of human GALR1 coding region, reverse primer.

[0226] KS-1311: 5′-CCT CAG TGA AGG GAA TGG GAG CGA-3′ (Seq. I.D. No. 3)Nucleotides 21-44 of rat GALR1 coding region, forward primer.

[0227] KS-1313: 5′-CTC ATT GCA AAC ACG GCA CTT GAA CA-3′ (Seq. I.D. No.4) Nucleotides 944-969 of rat GALR1 coding region, reverse primer.

[0228] KS-1447: 5′-CTT GCT TGT ACG CCT TCC GGA AGT-3′ (Seq. I.D. No. 5)Nucleotides 920-943 of rat GALR1 coding region, reverse primer.

[0229] KS-1448: 5′-GAG AAC TTC ATC ACG CTG GTG GTG-3′ (Seq. I.D. No. 6).Nucleotides 91-114 of rat GALR1 coding region, forward primer.

[0230] Generation of Human GALR2 PCR Product

[0231] Human genomic DNA (1 μg; 12 different lots from Promega andClontech) were amplified in 50 μl PCR reaction mixtures using the ExpandLong Template PCR System (as supplied and described by the manufacturer,Boehringer Mannheim) and 1 μM of primers, using a program consisting of40 cycles of 94° C. for 2 min, 60° C. for 2 min, and 68° C. for 3 min,with a pre- and post-incubation of 95° C. for 5 min and 68° C. for 10min, respectively. PCR primers for hGALR2 were designed against rGALR2sequence: forward primer NS525 in the fourth transmembrane domain, andreverse primer NS526 in the sixth transmembrane domain. The PCR productswere run on a 0.8% low-melting agarose gel. The single ≈300 bp fragmentfrom 3 different lots were isolated, purified by phenol extraction andsubjected to sequencing using the T7 Sequenase PCR product sequencingkit (Amersham). Sequence was analyzed using the Wisconsin Package (GCG,Genetics Computer Group, Madison, Wis.).

[0232] 5′ and 3′ RACE Analysis of Human GALR2

[0233] 5′ and 3′ RACE (rapid analysis of cDNA ends) were performed onhuman brain and human lung RNAs (Clontech), respectively, using aMarathon cDNA Amplification Kit (Clontech). Total RNA was poly A⁺selected using a FastTrack mRNA Isolation Kit (Invitrogen Corp., SanDiego, Calif.). For 5′ RACE, double stranded (ds) cDNA was synthesizedfrom 1 μg Poly A+ RNA using BB 153, a reverse primer from the 5′ end ofthe sixth transmembrane domain of hGALR2 from the PCR fragment describedabove. Adaptor ligation and nested PCR were performed according to theMarathon cDNA Amplification protocol using Advantage KlenTaq Polymerase(Clontech). The initial PCR reaction was performed on 1 μl of a 50 folddilution of the ligated cDNA using the supplier's Adaptor Primer 1 andBB 154, a reverse primer from the fifth transmembrane domain of thehGALR2 PCR product above. One μl of this initial PCR reaction wasre-amplified using the Adaptor Primer 2 and NS 563, a reverse primerjust upstream from BB154. The conditions for PCR were 30 sec at 94° C.,4 min at 72° C. for 5 cycles, 30 sec at 94° C., 4 min at 70° C. for 5cycles, 20 sec at 94° C., 4 min at 68° C. for 25 cycles, with a pre- andpost-incubation of 1 min at 94° C. and 7 min at 68° C. respectively. A600 base pair fragment from the nested PCR was isolated from a 1% TAEgel using a GENECLEAN III kit (BIO 101, Vista, Calif.) and sequencedusing AmpliTaq DNA Polymerase, FS (Perkin Elmer). The sequence was runon an ABI PRISM 377 DNA Sequencer and analyzed using the WisconsinPackage (GCG, Genetics Computer Group, Madison, Wis.). For 3′ RACE,double stranded (ds) cDNA was synthesized from 1 μg Poly A+ RNA usingthe cDNA synthesis primer CDS supplied with the Marathon cDNAAmplification Kit (Clontech). PCR conditions for 3′ RACE were similar to5′ RACE except that BB166 and BB167, forward primers from the fifthtransmembrane domain of the hGALR2 PCR fragment described above, wereused in place of BB154 and NS563, respectively. A 500 base pair fragmentfrom the nested PCR was isolated from a 1% TAE gel using a GENECLEAN IIIkit (BIO 101, Vista, Calif.) and sequenced as above.

[0234] Construction and Screening of a Human Heart cDNA Library

[0235] Poly A+ RNA was purified from human heart RNA (Clontech) using aFastTrack kit (Invitrogen, Corp.). DS− cDNA was synthesized from 8 μg ofpoly A+ RNA according to Gubler and Hoffman (1983) with minormodifications. The resulting cDNA was ligated to BstXI adaptors(Invitrogen, Corp.) and the excess adaptors removed by exclusion columnchromatography. High molecular weight fractions of size-selected ds-cDNAwere ligated in pEXJ.BS, an Okayama and Berg expression vector modifiedfrom pcEXV (Miller and Germain, 1986) to contain BstXI and otheradditional restriction sites. A total of 4.45×10⁶ independent cloneswith a mean insert size of 2.5 kb were generated. The library was platedon agar plates (Ampicillin selection) in 127 primary pools; 50 poolswith 37,500 independent clones, 51 pools with 25,000 clones and 26 poolswith 50,000 clones. Glycerol stocks of the primary pools were combinedin 16 superpools of 8 and screened for hGlR2 by PCR using primers BB153and BB169, a forward primer from the second intracellular domain ofhGALR2 identified in the 5′ RACE fragment above. PCR was performed withthe Expand Long Template PCR System (Boehringer Mannheim) under thefollowing conditions: 1 min at 94° C., 4 min at 68° C. for 40 cycles,with a pre- and post-incubation of 5 min at 95° C. and 7 min at 68° C.,respectively. Primary pools from positive superpools were screened byPCR and then primary pool 169 was subdivided and screened by PCR. Onepositive subpool, 69-11, was subdivided into 20 pools of 1200 clonesplated on agar plates (ampicillin selection). Colonies were transferredto nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.),denatured in 0.4 N NaOH, 1.5 M NaCl, renatured in 1M Tris, 1.5 M NaCl,and UV cross-linked. Filters were hybridized overnight at 40° C. in abuffer containing 50% formamide, 5×SSC, 7 mM TRIS, 1×Denhardt's solutionand 25 μg/ml salmon sperm DNA (Sigma Chemical Co.) and 10⁶ cpm/ml ofKS1567, an oligonucleotide probe from the 3′ end of the fifthtransmembrane domain of hGALR2, labeled with γ-³²P[ATP] (6000 Ci/mmol,NEN) using polynucleotide kinase (Boehringer Mannheim). Filters werewashed 2×15 minutes at room temperature in 2×SSC, 0.1% SDS, 2×15 minutesat 50° C. in 0.1×SSC, 0.1% SDS, and exposed to XAR X-ray film (Kodak)for 3 days. Colonies which appeared to hybridize were re-screened by PCRusing primers BB167 and BB170, a reverse primer from the COOH terminusof hGlR2 identified by the 3′ RACE fragment above. PCR was performedwith the Expand Long Template PCR System (Boehringer Mannheim) under thefollowing conditions: 1 min at 94° C., 2 min at 58° C., 2 min at 68° C.for 28 cycles, with a pre- and post-incubation of 5 min at 95° C. and 7min at 68° C. respectively. One positive colony, 69-11-5 was amplifiedovernight in 10 ml LB media and processed for plasmid purification usinga standard alkaline lysis miniprep procedure followed by a PEGprecipitation. To ensure that 69-11-5 was a single colony, it wasamplified for 3 hours in 3 ml of LB media and then 1 μl of a 1:100dilution was plated on an agar plate. Twenty colonies were screened byPCR using primers BB167 and BB170 using the same conditions as above,except that 25 cycles were used instead of 28. One positive singlecolony, 69-11-5-3, designated BO29, was amplified overnight in 10 ml ofTB media and processed for plasmid purification. Vector-anchored PCR wasperformed on BO29 using the Expand Long Template PCR System (BoehringerMannheim) to determine the orientation and size of the insert. BB173 andBB172, forward and reverse vector primers, respectively, were used withprimers BB169 and BB153. The conditions for PCR were 1 min at 94° C., 4min at 68° C. for 36 cycles, with a pre- and post-incubation of 5 min at95° C. and 7 min at 68° C. respectively. BO29 is preferably sequenced onboth strands using AmpliTaq DNA Polymerase, FS (Perkin Elmer). Thesequence is run on an ABI PRISM 377 DNA Sequencer and analyzed using theWisconsin Package (GCG, Genetics Computer Group, Madison, Wis.).

[0236] To test the ability of 69-11-5 to confer galanin binding, COS-7cells were plated in slide chambers (Lab-Tek) in Dulbecco's modifiedEagle medium (DMEM) supplemented with 10% calf serum, 100 U/ml ofpenicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (DMEM-c) and grownat 37° C. in a humidified 5% CO₂ atmosphere for 24 hours beforetransfection. Cells were transfected with 1 μg of miniprep DNA from69-11-5 or vector control using a modification of the DEAE-dextranmethod (Warden and Thorne, 1968). 48 hours after transfection, cellswere rinsed with phosphate-buffered saline (PBS) then incubated with 1nM ¹²⁵I -rat galanin (NEN; specific activity ˜2200 Ci/mmol) and 2 nM¹²⁵I-porcine galanin (NEN; specific activity ˜2200 Ci/mmol) in 20 mMHEPES-NaOH, pH 7.4, containing 1.26 mM CaCl₂, 0.81 mM MgS₄O, 0.44 mMKH₂PO₄, 5.4 mM KCl, 10 mM NaCl, 0.1% BSA, and 0.1% bacitracin for onehour at room temperature. After rinsing and fixation in 2.5%glutaraldehyde, slides were rinsed in PBS, air-dried, and dipped inphotoemulsion (Kodak, NTB-2). After a 4-day exposure, slides weredeveloped in Kodak D19 developer, fixed, and coverslipped (Aqua-Mount,Lerner Laboratories), then inspected for positive cells by brightfieldmicroscopy (Leitz Laborlux, 25× magnification). To test the ability ofthe single clone BO29 to confer galanin binding, BO29 or control vectorwere transfected into COS-7 cells for testing of ¹²⁵I galanin asdescribed above, with the exception that after fixation, binding of ¹²⁵Igalanin to cells on the slide was detected using an ¹²⁵I probe(Mini-Instruments, Ltd., Essex, England). The signal from BO29transfected cells was compared with the signal from control vectortransfected cells.

[0237] Primers and Probes Used NS525: 5′CCCTACCTGAGCTACTACCCTCA 3′; (SEQID NO:15) NS526: 5′ACCAAACCACACGCAGAGGATAAG 3′; (SEQ ID NO:16) BB153:5′-CCACGATGAGGATCATGCGTGTCACC-3′; (SEQ ID NO:17) BB154:5′-TAGGTCAGGCCGAGAACCAGCACAGG-3′; (SEQ ID NO:18) NS563:5′-CAGGTAGCTGAAGACGAAGGTGCA-3′; (SEQ ID NO:19) BB166:5′-CTGCACCTTCGTCTTCAGCTACCTG-3′; (SEQ ID NO:20) BB167:5′-CCTGTGCTGGTTCTCGCCCTGACCTA-3′; (SEQ ID NO:21) BB169:5′-TATCTGGCCATCCGCTACCCGCTGCA-3′; (SEQ ID NO:22) KS51567:5′-TTGCGCTACCTCTGGCGCGCCGTCGACCCGGTGGCCGCGGGCTCG-3′; (SEQ ID NO:23)BB170: 5′-CCAACAATGACTCCAACTCTGTGAC-3′; (SEQ ID NO:24) BB173:5′-AGGCGCAGAACTGGTAGGTATGGAA-3′; (SEQ ID NO:25) and BB172:5′-AAGCTTCTAGAGATCCCTCGACCTC-3′. (SEQ ID NO:26)

[0238] Generation of an Intronless Human GALR2 Receptor

[0239] Human tissues may be screened by PCR, using primers that crossthe intron, to identify cDNA sources that express the intronless form.An intronless hGALR2 clone may be obtained using an approach similar tothat used to obtain an intronless rGALR2 clone (infra). Alternatively,one may use restriction enzymes to remove the intron and some adjacentcoding region from BO29, and then replace the removed coding region byinserting a restriction enzyme-digested PCR fragment amplified from atissue shown to express the intronless form of the receptor.

[0240] Human hippocampus and human hypothalamus were each shown toexpress the intronless form. A full-length, intronless human GALR2 PCRproduct was amplified from human hippocampus, but was found to contain asingle point mutation downstream from the intron splice site. Therefore,an EcoRI/StyI restriction digest fragment, containing 11 bp of 5′UT andthe first 557 bp of hGalR2 coding region, was ligated to a StyIrestriction digest fragment, containing bp 558-1164 of the coding regionand 182 bp of 3′ UT, which was isolated from the intron-containinghGalR2 clone (BO29). The ligation product, comprising the entireintronless form of the human GALR2 receptor, was subcloned into thevector pEXJ and designated BO39.

[0241] Northern Blots

[0242] Human brain multiple tissue northern blots (MTN blots II and III,Clontech, Palo Alto, Calif.) carrying mRNA purified from various humanbrain areas may be hybridized according to the manufacturers'specifications.

[0243] Rat multiple tissue northern blots including multiple braintissue blots (rat MTN blot, Clontech, Palo Alto, Calif.) carrying mRNApurified from various rat tissues also may be hybridized at highstringency according to the manufacturer's specifications.

[0244] RT-PCR Analyses of GALR2 mRNA

[0245] Tissues may be homogenized and total RNA extracted using theguanidine isothiocyanate/CsCl cushion method. RNA may then be treatedwith DNase to remove any contaminating genomic DNA. cDNA may be preparedfrom total RNA with random hexanucleotide primers using the reversetranscriptase Superscript II (BRL, Gaithersburg, Md.). First strand cDNA(about 250 ng of total RNA) may be amplified for example, in a 50 μL PCRreaction mixture (200 μM dNTPs final concentration) and 1 μM appropriateprimers, using an appropriate thermal cycling program.

[0246] The PCR products may be run on a 1.5% agarose gel and transferredto charged nylon membranes (Zetaprobe GT, BioRad), and analyzed asSouthern blots. GALR2 primers will be screened for the absence ofcross-reactivity with the other galanin receptors. Filters may behybridized with radiolabeled probes and washed under high stringency.Labeled PCR products may be visualized on X-ray film. Similar PCR andSouthern blot analyses may be conducted with primers and probes, e.g.,1B15, directed to the housekeeping gene, glyceraldehyde phosphatedehydrogenase (Clontech, Palo Alto, Calif.), to normalize the amount ofcDNA used from the different tissues.

[0247] RT PCR of rat brain tissues was carried out using total or polyA⁺ RNA (1.5 μg or 0.5 μg, respectively) isolated from various rat brainregions and converted to cDNA using Superscript II (BRL, Gaithersburg,Md.) reverse transcriptase with random priming. The cDNAs were used astemplates for PCR amplification of GALR2 using specific GALR2 primers.PCR products were separated on an agarose gel by electrophoresis andblotted to a charged nylon membrane.

[0248] Isolation of the Intronless Rat GALR2

[0249] RT-PCR analysis of various rat brain regions (FIG. 5) was carriedout using primers representing N- and C-termini of rat GALR2 (supra).The forward and reverse primers comprised nucleotides 1-23 and1087-1110, respectively, of the intronless rat GALR2 sequence (SEQ. IDNo. 7). The PCR products were separated by agarose gel electrophoresis,blotted, and hybridized with an oligonucleotide probe designed to thepredicted 5/6 loop of GALR2 (nucleotides 651-695, SEQ. ID No. 7). Thisanalysis indicated the presence of both intron-containing and intronlessforms of rat GALR2 in brain. In order to choose an appropriate tissuesource from which to isolate the intronless form, a similar PCR analysison RNA from a variety of rat tissues was carried out. Based on the sizeof the products determined by agarose gel electrophoresis (data notshown), rat heart was chosen as a potential source of intronless GALR2RNA. To isolate the intronless GALR2, PCR primers similar to those usedabove but containing restriction enzyme sites to facilitate subcloningand a Kozak consensus for translation initiation (KS-1550 and KS-1551,see below) were used to amplify rat GALR2 from rat heart RNA by PCR(after conversion of the RNA to first strand cDNA by standard methods).A PCR product of the correct size was isolated from an agarose gel andthen reamplified using the same primers to increase yield. The productswere digested with the appropriate restriction enzymes to producecohesive ends (EcoRI and Xba I), ligated into the expression vectorEXJ.RH and transformed into E.coli. The resulting colonies weretransferred to nitrocellulose membranes and hybridized with anoligonucleotide probe to the predicted 2/3 loop of rat GALR2(nucleotides 259-303, SEQ. ID No. 7). A single hybridizing colony wasfound by subsequent analysis to contain the intronless rat GALR2 cDNA.

[0250] Primers used: Forward primer, KS-1550: (SEQ. ID No.27)5′-ACGGAATTCGACATGAATGGCTCCGGCA Reverse Primer, KS-1551: (SEQ. ID No.28)5′-GCTCTAGAGCCCCTTTGGTCCTTTAACAAGCCGG

[0251] Production of Recombinant Baculovirus

[0252] The coding region of GALR2 may be subcloned into pBlueBacIII intoexisting restriction sites, or sites engineered into sequences 5′ and 3′to the coding region of GALR2, for example, a 5′ BamHI site and a 3′EcoRI site. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of GALR2 construct may be co-transfected into 2×10⁶ Spodopterafrugiperda insect Sf9 cells by the calcium phosphate co-precipitationmethod, as outlined in by Pharmingen (in “Baculovirus Expression VectorSystem: Procedures and Methods Manual”). The cells then are incubatedfor 5 days at 27° C.

[0253] The supernatant of the co-transfection plate may be collected bycentrifugation and the recombinant virus plaque purified. The procedureto infect cells with virus, to prepare stocks of virus and to titer thevirus stocks are as described in Pharmingen's manual.

[0254] Cell Culture

[0255] COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/mL penicillin/100 μg/mL streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days. Human embryonic kidney 293 cells are grown on 150 mm plates inD-MEM with supplements (minimal essential medium) with Hanks' salts andsupplements (Dulbecco's Modified Eagle Medium with 10% bovine calfserum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mL streptomycin)at 37° C., 5% CO₂. Stock plates of 293 cells are trypsinized and split1:6 every 3-4 days. Mouse fibroblast LM(tk−) cells are grown on 150 mmplates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin) at 37° C., 5% CO₂. Stock plates of LM(tk−) cells aretrypsinized and split 1:10 every 3-4 days. Chinese hamster ovary (CHO)cells were grown on 150 mm plates in HAM's F-12 medium with supplements(10% bovine calf serum, 4 mM L-glutamine and 100 units/mL penicillin/100ug/ml streptomycin) at 37° C., 5% CO2. Stock plates of CHO cells weretrypsinized and split 1:8 every 3-4 days.

[0256] LM(tk−) cells stably transfected with the GALR2 receptor may beroutinely converted from an adherent monolayer to a viable suspension.Adherent cells are harvested with trypsin at the point of confluence,resuspended in a minimal volume of complete DMEM for a cell count, andfurther diluted to a concentration of 10⁶ cells/mL in suspension media(10% bovine calf serum, 10% 10×Medium 199 (Gibco), 9 mM NaHCO₃, 25 mMglucose, 2 mM L-glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin, and 0.05% methyl cellulose). Cell suspensions aremaintained in a shaking incubator at 37° C., 5% CO₂ for 24 hours.Membranes harvested from cells grown in this manner may be stored aslarge, uniform batches in liquid nitrogen. Alternatively, cells may bereturned to adherent cell culture in complete DMEM by distribution into96-well microtiter plates coated with poly-D-lysine (0.01 mg/mL)followed by incubation at 37° C., 5% CO₂ for 24 hours. Cells prepared inthis manner generally yield a robust and reliable response in cAMPradio-immunoassays as further described hereinbelow.

[0257] Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mmplates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10%bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 μg/mLstreptomycin) at 37° C., 5% CO2. Stock plates of NIH-3T3 cells aretrypsinized and split 1:15 every 3-4 days.

[0258] Sf9 and Sf21 cells are grown in monolayers on 150 mm tissueculture dishes in TMN-FH media supplemented with 10% fetal calf serum,at 27° C., no CO₂. High Five insect cells are grown on 150 mm tissueculture dishes in Ex-Cell 400™ medium supplemented with L-Glutamine,also at 27° C., no CO₂.

[0259] Transfection

[0260] All receptor subtypes studied may be transiently transfected intoCOS-7 cells by the DEAE-dextran method, using 1 μg of DNA/10⁶ cells(Cullen, 1987). In addition, Schneider 2 Drosophila cells may becotransfected with vectors containing the receptor gene, under controlof a promoter which is active in insect cells, and a selectableresistance gene, eg., the G418 resistant neomycin gene, for expressionof the galanin receptor.

[0261] Stable Transfection

[0262] The GALR2 receptor may be co-transfected with a G-418 resistantgene into the human embryonic kidney 293 cell line by a calciumphosphate transfection method (Cullen, 1987). GALR1 receptors wereexpressed in cells using methods well-known in the art. Stablytransfected cells are selected with G-418. GALR2 receptors may besimilarly transfected into mouse fibroblast LM(tk−) cells, Chinesehamster ovary (CHO) cells and NIH-3T3 cells. Transfection of LM(tk−)cells with the plasmid K985 and subsequent selection with G-418 resultedin the LM(tk−) cell line L-rGALR2-8 (ATCC Accession No. CRL-12074),which stably expresses the rat GALR2 receptor. A similar procedure wasused to transfect LM(tk−) cells with plasmid K1045 (intronless rat GALR2receptor construct) resulting in the LM(tk−) cell line L-rGALR4-I (ATCCAccession No. CRL-12223). In addition, this procedure was used totransfect CHO cells with an intron-containing plasmid to create a stablyexpressing rat GALR2 CHO cell line, C-GalR2-79 (ATCC Accession No.______).

[0263] Radioligand Binding Assays

[0264] Transfected cells from culture flasks were scraped into 5 ml ofTris-HCl, 5mM EDTA, pH 7.5, and lysed by sonication. The cell lysateswere centrifuged at 1000 rpm for 5 min. at 4° C., and the supernatantwas centrifuged at 30,000×g for 20 min. at 4° C. The pellet wassuspended in binding buffer (50 mM Tris-HCl, 5 mM MgSO₄, 1 mM EDTA at pH7.5 supplemented with 0.1% BSA, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin,and 10 μg/ml phosphoramidon). Optimal membrane suspension dilutions,defined as the protein concentration required to bind less than 10% ofthe added radioligand, were added to 96-well polpropylene microtiterplates containing ¹²⁵I-labeled peptide, non-labeled peptides and bindingbuffer to a final volume of 250 μl. In equilibrium saturation bindingassays membrane preparations were incubated in the presence ofincreasing concentrations (0.1 nM to 4 nM) of [¹²⁵I]porcine galanin(specific activity 2200 Ci/mmol).

[0265] The binding affinities of the different galanin analogs weredetermined in equilibrium competition binding assays, using 0.1 nM[¹²⁵I]porcine galanin in the presence of twelve different concentrationsof the displacing ligands. Binding reaction mixtures were incubated for1 hr at 30° C., and the reaction was stopped by filtration through GF/Bfilters treated with 0.5% polyethyleneimine, using a cell harvester.Radioactivity was measured by scintillation counting and data wereanalyzed by a computerized non-linear regression program. Non-specificbinding was defined as the amount of radioactivity remaining afterincubation of membrane protein in the presence of 100 nM of unlabeledporcine galanin. Protein concentration was measured by the Bradfordmethod using Bio-Rad Reagent, with bovine serum albumin as a standard.

[0266] Binding assays involving the rat GALR3 receptor are conducted atroom temperature for 120 min. in binding buffer. Leupeptin, aprotoninand phosphoramidon are omitted from rat GALR3 assays while bacitracin isadded to 0.1%. Nonspecific binding is defined in the presence of 1 μMporcine galanin. Cells transiently or stably expressing GALR3 receptorsare produced using transfection methods which are well-known in the art,examples of which are provided herein (supra). The rat GALR3 receptormay be expressed using plasmid K1086, deposited on Oct. 8, 1996, withthe ATCC, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A under theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure, and was accordedATCC Accession No. 97747. Another plasmid expressing the rat GALR3receptor is plasmid pEXJ-rGALR3t, deposited with the ATCC under theBudapest Treaty on Dec. 17, 1996, and accorded ATCC Accession No. 97826.The human GALR3 receptor may be expressed using plasmid pEXJ-hGALR3,also deposited with the ATCC under the Budapest Treaty on Dec. 17, 1996,and accorded ATCC Accession No. 97827. Cells stably expressing the GALR3receptors may be used in functional assays well known in the art,examples of which are provided herein (infra).

[0267] Functional Assays

[0268] Cyclic AMP (cAMP) Formation

[0269] The receptor-mediated inhibition of cyclic AMP (cAMP) formationmay be assayed in LM(tk−) cells expressing the rat GALR1 and GALR2receptors. Cells were plated in 96-well plates and incubated inDulbecco's phosphate buffered saline (PBS) supplemented with 10 mMHEPES, 5 mM theophylline, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10μg/ml phosphoramidon for 20 min at 37° C., in 5% CO₂. Galanin or thetest compounds were added and incubated for an additional 10 min at 37°C. The medium was aspirated and the reaction was stopped by the additionof 100 mM HCl. The plates were stored at 4° C. for 15 min, and the cAMPcontent in the stopping solution was measured by radioimmunoassay.Radioactivity was quantified using a gamma counter equipped with datareduction software.

[0270] Functional assay experiments were also performed using stablytransfected cells seeded into 96-well microtiter plates and cultureduntil confluent. To reduce the potential for receptor desensitization,the serum component of the media was reduced to 1.5% for 4 to 16 hoursbefore the assay. Cells were washed in Hank's buffered saline, or HBS(150 mM NaCl, 20 mM HEPES, 1 mM CaCl₂, 5 mM KCl, 1 mM Mg₂Cl, and 10 mMglucose) supplemented with 0.1% bovine serum albumin plus 5 mMtheophylline and pre-equilibrated in the same solution for 20 min at 37°C. in 5% CO₂. Cells were then incubated 5 min with 10 μM forskolin andvarious concentrations of receptor-selective ligands. The assay wasterminated by the removal of HBS and acidification of the cells with 100mM HCl. Intracellular cAMP was extracted and quantified with a modifiedversion of a magnetic bead-based radioimmunoassay (Advanced Magnetics,Cambridge, Mass.). The final antigen/antibody complex was separated fromfree ¹²⁵I-cAMP by vacuum filtration through a PVDF filter in amicrotiter plate (Millipore, Bedford, Mass.). Filters were punched andcounted for ¹²⁵I in a Packard gamma counter. Functional studies of therat GALR1 receptor in LMTK− cells were performed as previously describedabove except that leupeptin, aprotinin and phosphoramidon were omittedfrom the assay, and cells were stimulated with forskolin plus peptidesfor a period of 5 min.

[0271] Arachidonic Acid Release

[0272] CHO cells stably transfected with the rat GALR2 receptor wereseeded into 96 well plates and grown for 3 days in HAM's F-12 withsupplements. ³H-arachidonic acid (specific activity=0.75 uCi/ml) wasdelivered as a 100 ul aliquot to each well and samples were incubated at37° C., 5% CO₂ for 18 hours. The labeled cells were washed three timeswith 200 ul HAM's F-12. The wells were then filled with medium (200 uL)and the assay was initiated with the addition of peptides or buffer (22uL). Cells were incubated for 30 min at 37° C., 5% CO2. Supernatantswere transferred to a microtiter plate and evaporated to dryness at 75°C. in a vacuum oven. Samples were then dissolved and resuspended in 25uL distilled water. Scintillant (300 uL) was added to each well andsamples were counted for ³H in a Trilux plate reader. Data were analyzedusing nonlinear regression and statistical techniques available in theGraphPAD Prism package (San Diego, Calif.).

[0273] Intracellular Calcium Mobilization

[0274] The intracellular free calcium concentration may be measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(Bush et al. 1991). Cells stably transfected with GALR2 are seeded ontoa 35 mm culture dish containing a glass coverslip insert. Cells arewashed with HBS and loaded with 100 μL of Fura-2/AM (10 μM) for 20 to 40min. After washing with HBS to remove the Fura-2/AM solution, cells areequilibrated in HBS for 10 to 20 min. Cells are then visualized underthe 40× objective of a Leitz Fluovert FS microscope and fluorescenceemission is determined at 510 nM with excitation wavelengths alternatingbetween 340 nM and 380 nM. Raw fluorescence data are converted tocalcium concentrations using standard calcium concentration curves andsoftware analysis techniques.

[0275] Phosphoinositide Metabolism

[0276] LM(tk−) cells stably expressing the rat GALR2 receptor cDNA wereplated in 96-well plates and grown to confluence. The day before theassay the growth medium was changed to 100 μl of medium containing 1%serum and 0.5 μCi [³H]myo-inositol, and the plates were incubatedovernight in a CO₂ incubator (5%₂ CO at 37° C.). Alternatively,arachidonic acid release may be measured if [³H]arachidonic acid issubstituted for the ³[H]myo-inositol. Immediately before the assay, themedium was removed and replaced by 200 μL of PBS containing 10 mM LiCl,and the cells were equilibrated with the new medium for 20 min. Duringthis interval cells were also equilibrated with the antagonist, added asa 10 μL aliquot of a 20-fold concentrated solution in PBS. The[³H]inositol-phosphates accumulation from inositol phospholipidmetabolism was started by adding 10 μL of a solution containing theagonist. To the first well 10 μL were added to measure basalaccumulation, and 11 different concentrations of agonist were assayed inthe following 11 wells of each plate row. All assays were performed induplicate by repeating the same additions in two consecutive plate rows.The plates were incubated in a CO₂ incubator for 1 hr. The reaction wasterminated by adding 15 μl of 50% v/v trichloroacetic acid (TCA),followed by a 40 min. incubation at 4° C. After neutralizing TCA with 40μl of 1M Tris, the content of the wells was transferred to a MultiscreenHV filter plate (Millipore) containing Dowex AG1-X8 (200-400 mesh,formate form). The filter plates were prepared adding 200 μL of DowexAG1-X8 suspension (50% v/v, water:resin) to each well. The filter plateswere placed on a vacuum manifold to wash or elute the resin bed. Eachwell was washed 2 times with 200 μL of water, followed by 2×200 μL of 5mM sodium tetraborate/60 mM ammonium formate. The [³H]IPs were elutedinto empty 96-well plates with 200 μl of 1.2 M ammonium formate/0.1formic acid. The content of the wells was added to 3 mls ofscintillation cocktail, and the radioactivity was determined by liquidscintillation counting.

[0277] Functional assays using GALR3 receptors are performed similarly.

[0278] It is to be understood that the cell lines described herein aremerely illustrative of the methods used to evaluate the binding andfunction of the galanin receptors of the present invention, and thatother suitable cells may be used in the assays described herein.

[0279] Functional Responses in Oocytes Expressing GalR2

[0280] Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) wereanesthetized in 0.2% tricain (3-aminobenzoic acid ethyl ester, SigmaChemical Corp.) and a portion of ovary was removed using aseptictechnique (Quick and Lester, 1994). Oocytes were defolliculated using 2mg/ml collagenase (Worthington Biochemical Corp., Freehold, N.J.) in asolution containing 87.5 mM NaCl, 2 mM KCl, 2 mM MgCl₂ and 5 mM HEPES,pH 7.5. oocytes were injected (Nanoject, Drummond Scientific, Broomall,Pa.) with 50 nL of rat GalR2 mRNA or other mRNA for use as a negativecontrol. RNA was prepared by linearization of the plasmid (pBluescript)containing the entire coding region of the GalR2 cDNA, followed by invitro transcription using the T7 polymerase (“MessageMachine”, Ambion).Alternatively, mRNA may be translated from a template generated by PCR,incorporating a T7 promoter. Oocytes were incubated at 16° on a rotatingplatform for 3-8 days post-injection. Dual electrode voltage clamp(“GeneClamp”, Axon Instruments Inc., Foster City, Calif.) was performedusing 3 M KCl-filled glass microelectrodes having resistances of 1-3Mohms. Unless otherwise specified, oocytes were clamped at a holdingpotential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (2-5 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 2 mM CaCl₂, 2 mM MgCl₂, 5 mM HEPES, pH 7.5 (ND96). Drugs areapplied by switching from a series of gravity fed perfusion lines.

[0281] The human GALR2 receptor and GALR3 receptors may be studiedfunctionally using similar methods.

[0282] Galanin Receptor Autoradiography

[0283] Male Sprague-Dawley rats (Charles River, Wilmington, Mass.) wereeuthanized using Co₂, decapitated, and their brains immediately removedand frozen on dry ice. Tissue sections were cut at 20 μm using acryostat and thaw mounted onto gelatin coated slides. Tissues werepreincubated in two 10 minute changes of 50 mM Tris-HCl buffer pH 7.4,containing 5 mM MgSO₄ and 2 mM EGTA (Sigma). The radioligand binding wascarried out in the same buffer, which also contained 0.1% bovine serumalbumin, 0.02% aprotinin, 0.031% leupeptin, 0.1% phosphoramidate(Boehringer Mannheim), and 0.1 nM [¹²⁵I]porcine galanin (specificactivity 2200 Ci/mmol, NEN) for 1 hour at 22° C. Nonspecific binding wasdetermined in the presence of 5 μM porcine galanin (Bachem). As[D-Trp²]galanin₍₁₋₂₉₎ was shown to be selective for the cloned GAlR2receptor (infra), a 60 nM concentration of this peptide was used todisplace [¹²⁵I]galanin binding from the rat brain tissue sections. Theuse of this concentration was based on the binding data, which showedthe affinity of [D-Trp²]galanin₍₁₋₂₉₎ to be 6 nM at the GALR2 receptor,and 3 μM at the GALR1 receptor. In general, a 10×concentration of theblocking ligand is sufficient to remove 100% of the targeted receptor,while leaving the GALR1 receptor unaffected. After incubation, tissueswere dipped twice in ice-cold Tris-HCl buffer (4° C.), followed by a 5minute wash in ice-cold Tris-HCl buffer (4° C.), then dipped twice inice-cold deionized water to remove the salts. Sections were placed inX-ray cassettes and apposed to Dupont Cronex MRF 34 Film for 5 days.Films were developed using a Kodak M35A Processor.

[0284] Tissue Preparation for Neuroanatomical Studies

[0285] Male Sprague-Dawley rats (Charles River) are decapitated and thebrains rapidly removed and frozen in isopentane. Coronal sections arecut at 11 μm on a cryostat and thaw-mounted onto poly-L-lysine coatedslides and stored at −80° C. until use. Prior to hybridization, tissuesare fixed in 4% paraformaldehyde, treated with 5 mM dithiothreitol,acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride,delipidated with chloroform, and dehydrated in graded ethanols.

[0286] Probes

[0287] oligonucleotide probes employed to characterize the distributionof the rat GALR2 receptor mRNA may be synthesized, for example, on aMillipore Expedite 8909 Nucleic Acid Synthesis System. The probes arethen lyophilized, reconstituted in sterile water, and purified on a 12%polyacrylamide denaturing gel. The purified probes are againreconstituted to a concentration of 100 ng/μL, and stored at −20° C.Probe sequences may include DNA or RNA which is complementary to themRNA which encodes the GALR2 receptor.

[0288] Localization of GALR2 mRNA: In situ Hybridization Animals

[0289] Timed-pregnant female Sprague-Dawley rats were puchased fromCharles River. The day of birth for each litter was designated aspostnatal day 0 (P0). Brains were removed from pups on P0, P3, P5, P8,P10, P15, P20, and P25. The brains from the mothers were also removedand used as the adult comparison. All brains were sectioned in thecoronal plane at 11 μm and the sections thaw-mounted on to poly-1-lysinecoated microscope slides. The sections were then used for in situhybridization histochemistry as described below.

[0290] Tissue Preparation

[0291] Prior to hybridization, tissues were fixed in 4%paraformaldehyde, treated with 5 mM dithiothreitol, acetylated in 0.1 Mtriethanolamine containing 0.25% acetic anhydride, delipidated withchloroform, and dehydrated in graded ethanols. The sections wereprehybridized for one hour at 40° C. in hybridization buffer, whichconsisted of 50% formamide, 4×sodium citrate buffer (1×SSC=0.15 M NaCland 0.015 M sodium citrate), 1×Denhardt's solution (0.2%polyvinylpyrrolidine, 0.2% Ficoll, 0.2% bovine serum albumin), 50 mMdithiothreitol, 0.5 mg/ml salmon sperm DNA, 0.5 mg/ml yeast tRNA, and10% dextran sulfate.

[0292] In Situ Hybridization

[0293] 32mer oligonucleotide probes complementary to nucleotides 261-292of the GALR2 mRNA were synthesized, purified, and 3′-end labeled with³⁵S-dATP (1200 Ci/mmol, New England Nuclear, Boston, Mass.) to aspecific activity of 10⁹ dpm/μg using terminal deoxynucleotidyltransferase (Boehringer Mannheim; Indianapolis, Ind.). The radiolabeledprobes were purified on Biospin 6 chromatography columns (Bio-Rad;Richmond, Calif.), and diluted in the hybridization buffer describedabove to a concentration of 1.5×10⁴ cpm/μl. One hundred μl of theradiolabeled probe was applied to each section, which was then coveredwith a Parafilm coverslip. Hybridization was carried out overnight inhumid chambers at 40 to 55° C. The following day the sections werewashed in two changes of 2×SSC for one hour at room temperature, in2×SSC for 30 min at 50-60° C., and finally in 0.1×SSC for 30 min at roomtemperature. Tissues were dehydrated in graded ethanols and apposed toKodak XAR-5 film for 2 weeks at −20° C., then dipped in Kodak NTB3autoradiography emulsion diluted 1:1 with 0.2% glycerol water. Afterexposure at 4° C. for 4 weeks, the slides were developed in Kodak D-19developer, fixed, and counterstained with hematoxylin and eosin.

[0294] Localization of GALR2 mRNA: Ribonuclease Protection Assay (RPA)

[0295] Development of Probes

[0296] A cDNA fragment encoding a 467 BP fragment of the rGAL R2 wassubcloned into a pBluescript plasmid vector. This construct waslinearized with Xba I or Sal I. T3 and T7 RNA polymerases were used tosynthesize the sense and antisense strands of RNA respectively.Full-length RNA transcripts were obtained using a full-length cDNAconstruct in the same vector.

[0297] A probe coding for rat glyceraldehyde 3-phosphate dehydrogenase(GAPDH) gene, a constitutively expressed protein, was used concurrently.GAPDH is expressed at a relatively constant level in most tissue and itsdetection was used to compare expression levels of the rGalR2 gene indifferent tissues.

[0298] RNA Extraction

[0299] RNA was isolated from rat peripheral tissue as well as regions ofthe CNS using a LiCl precipitation protocol (Cathala et al.,. 1983).Tissue was homogenized in 5M guanidine isothiocyanate, 50 mM TRIS, 10 mMEDTA, using 7 ml of lysis buffer/gram tissue. 4M LiCl were added (7ml/ml homogenate) and the mixture were stored at 4° C. for 24-48 hours.Homogenates were centrifuged and the pellets were resuspended in 3MLiCl, and centrifuged again. The pellets were resuspended in 0.1% sodiumdodecyl sulfate (SDS), extracted in phenol:chloroform:isoamyl alcohol(24:24:1) and the RNA ethanol precipitated. Yield and relative puritywere assessed by measuring absorbance A₂₆₀/A₂₈₀.

[0300] Synthesis of Probes

[0301] rGALR2 and GAPDH cDNA sequences preceded by phage polymerasepromoter sequences were used to synthesize radiolabeled riboprobes.Conditions for the synthesis of riboprobes were: 1-2 μl linearizedtemplate (1 μg/μl), 1 μl of ATP, GTP, UTP (10 mM each), 2 μldithiothreitol (0.1 M), 20 units RNAsin RNAse inhibitor, 1-2 μl (15-20units/μl) RNA polymerase, 4 μl transcription buffer (Promega Corp.), and5 μl α^(P)-CTP (specific activity 800 Ci/mmol). 0.1 mM CTP (0.02-1.0 μl)were added to the reactions, and the volume were adjusted to 20 μl withDEPC-treated water. Labeling reactions were incubated at 38° C. for 90min, after which 2 units of RQ1 RNAse-free DNAse (Promega Corp.) wereadded to digest the template. The riboprobes were separated fromunincorporated nucleotide by a spun G-50 column (Select D G-50(RF); 5Prime-3 Prime, Inc.). TCA precipitation and liquid scintillationspectrometry were used to measure the amount of label incorporated intothe probe. A fraction of all riboprobes synthesized weresize-fractionated on 0.4 mm thick 5% acrylamide sequencing gels andautoradiographed to confirm that the probes synthesized were full-lengthand not degraded.

[0302] Solution Hybridization/Ribonuclease Protection Assay

[0303] For solution hybridization 2-15 μg of total RNA isolated fromtissues were used. Sense RNA synthesized using the full-length codingsequence of the rGalR2 was used to characterize specific hybridization.Negative controls consisted of 30 μg transfer RNA (tRNA) or no tissueblanks. All samples were placed in 1.5-ml microfuge tubes and vacuumdried. Hybridization buffer (40 μl of 400 mM NaCl, 20 mM Tris, pH 6.4, 2mM EDTA, in 80% formamide) containing 0.25-1.0×10⁶ counts of each probewere added to each tube. Samples were heated at 90° C. for 15 min, afterwhich the temperature were lowered to 45° C. for hybridization.

[0304] After hybridization for 14-18 hr, the RNA/probe mixtures weredigested with RNAse A (Sigma) and RNAse T1 (Bethesda Research Labs). Amixture of 2.0 μg RNAse A and 1000 units of RNAse T1 in a buffercontaining 330 mM NaCl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 μl) wasadded to each sample and incubated for 90 min at room temperature. Afterdigestion with RNAses, 20 μl of 10% SDS and 50 μg proteinase K wereadded to each tube and incubated at 37° C. for 15 min. Samples were thenextracted with phenol/chloroform:isoamyl alcohol and precipitated in 2volumes of ethanol for 1 hr at −70° C. tRNA was added to each tube (30mg) as a carrier to facilitate precipitation. Following precipitation,samples were centrifuged, washed with cold 70% ethanol, and vacuumdried. Samples were dissolved in formamide loading buffer andsize-fractionated on a urea/acrylamide sequencing gel (7.6 M urea, 6%acrylamide in Tris-borate-EDTA). Gels were dried and apposed to KodakXAR-5 x-ray film.

[0305] In vivo Methods

[0306] The effects of galanin, galanin derivatives, and related peptidesand compounds were evaluated by intracerebroventricular (i.c.v.)injection of the peptide or compound followed by measurement of foodintake in the animal. Measurement of food intake was performed for 3hours after injection, but other protocols may also be used. Saline wasinjected as a control, but it is understood that other vehicles may berequired as controls for some peptides and compounds. In order todetermine whether a compound is a GALR2 antagonist, food intake in ratsmay be stimulated by administration of (for example) the GALR2-selectivepeptide agonist [D-Trp₂]-galanin₍₁₋₂₉₎ through anintracerebroventricular (i.c.v.) cannula. A preferred anatomic locationfor injection is the hypothalamus, in particular, the paraventricularnucleus. Methods of cannulation and food intake measurements arewell-known in the art, as are i.c.v. modes of administration (Kyrkouliet al., 1990, Ogren et al., 1992). To determine whether a compoundreduces [D-Trp₂]-galanin₍₁₋₂₉₎ stimulated food intake, the compound maybe administered either simultaneously with the peptide, or separately,either through cannula, or by subcutaneous, intramuscular, orintraperitoneal injection, or more preferably, orally.

[0307] Materials

[0308] Cell culture media and supplements are from Specialty Media(Lavallette, N.J.). Cell culture plates (150 mm and 96-well microtiter)are from Corning (Corning, N.Y.). Sf9, Sf21, and High Five insect cells,as well as the baculovirus transfer plasmid, pBlueBacIII™, are purchasedfrom Invitrogen (San Diego, Calif.). TMN-FH insect medium complementedwith 10% fetal calf serum, and the baculovirus DNA, BaculoGold™, isobtained from Pharmingen (San Diego, CA.). Ex-Cell 400™ medium withL-Glutamine is purchased from JRH Scientific. Polypropylene 96-wellmicrotiter plates are from Co-star (Cambridge, Mass.). All radioligandsare from New England Nuclear (Boston, Mass.). Galanin and relatedpeptide analogs were either from Bachem California (Torrance, Calif.),Peninsula (Belmont, Calif.); or were synthesized by custom order fromChiron Mimotopes Peptide Systems (San Diego, Calif.).

[0309] Bio-Rad Reagent was from Bio-Rad (Hercules, Calif.). Bovine serumalbumin (ultra-fat free, A-7511) was from Sigma (St. Louis. Mo.). Allother materials were reagent grade.

[0310] Experimental Results

[0311] Isolation of a GALR2 cDNA from Rat Hypothalamus

[0312] In order to clone additional members of the galanin receptorfamily, an expression cloning strategy based on the potential presenceof multiple galanin receptors in hypothalamus was designed. Althoughrecent evidence indicated that GALR1 receptor mRNA was present in rathypothalamus (Gustafson et al., 1996; Parker et al., 1995), not allaspects of the cloned GALR1 pharmacological profile match that observedfor galanin-mediated feeding (Crawley et al., 1993). These resultssuggested that the regulation of galanin-induced feeding may not beexplained by the presence of only GALR1 in the rat hypothalamus.

[0313] A randomly-primed cDNA expression library was constructed fromrat hypothalamus and screened by radioligand binding/photoemulsiondetection using [¹²⁵I]-porcine galanin. The library consisted of 584pools containing about 5,000 primary clones/pool for a total of about 3million clones with an average insert size of 2.2 kb. Pools positive forrat GALR1 (about 110) were eliminated from the screen. Remaining poolswere screened for radioligand binding using 1 nM [¹²⁵I]-porcine galanin;slides were inspected for positive cells by direct microscopicexamination. One positive pool (J126) was subdivided into 96 pools ofabout 90 clones each and rescreened for galanin binding. Preliminarypharmacology carried out on the positive subpool J126-10 indicated thatthe [¹²⁵I]-porcine galanin binding was not sensitive to inhibition bygalanin 3-29. 400 individual colonies of a positive pool (J26-10) werethen screened to find two single purified cDNA clones. J126-10-334 waschosen for further analysis and designated K985. PCR analysis usingthree independent GALR1 primer sets (see Methods; data not shown)confirmed that the newly isolated cDNA was distinct from GALR1 and thusencoded a new galanin receptor subtype, termed GALR2.

[0314] The isolated clone K985 carries a 3.8 kb insert. Sequenceanalysis of this cDNA revealed a complete coding region for a novelreceptor protein which we term GALR2 (see FIGS. 1 and 2). Searches ofGenEMBL databases indicated that the sequence was novel, and that themost similar sequence was that of the galanin receptor GALR1, followedby other G protein-coupled receptors (GPCR). The nucleotide and deducedamino acid sequences are shown in FIGS. 1 and 2, respectively. Thenucleotide sequence of the coding region is ˜56% identical to rat GALR1and ˜54% identical to human GALR1 and encodes a 372 amino acid proteinwith 38% and 40% amino acid identity to rat and human GALR1,respectively. Hydropathy plots of the predicted amino acid sequencereveal seven hydrophobic regions that may represent transmembranedomains (TMs, data not shown), typical of the G protein-coupled receptorsuperfamily. In the putative TM domains, GALR2 exhibits 48-49% aminoacid identity with rat and human GALR1. Like most GPCRs, the GALR2receptor contains consensus sequences for N-linked glycosylation in theN-terminus (positions 2 and 11) as well as the predicted extracellularloop between TMs IV and V. The GALR2 receptor contains two highlyconserved cysteine residues in the first two extracellular loops thatare believed to form a disulfide bond stabilizing the functional proteinstructure (Probst et al., 1992). GALR2 shows five potentialphosphorylation sites for protein kinase C in positions 138, 210, 227,319, and 364, and two cAMP- and cGMP-dependent protein kinasephosphorylation sites in positions 232 and 316. It should be noted thatsix out of the seven potential phosphorylation sites are located inpredicted intracellular domains, and therefore could play a role inregulating functional characteristics of the GALR2 receptor (Probst etal., 1992).

[0315] Within the GALR2 cDNA K985 (J126-10-334) isolated from the rathypothalamus library, the coding region of GALR2 is interrupted by anintron of ˜1 kb (FIGS. 3A, 3B, and 3C). A cDNA containing an intron maybe produced by the action of reverse transcriptase on an incompletelyspliced form of messenger RNA. The heterologous expression of thecomplete protein product is not necessarily impeded by the presence ofthe intron in the coding region, because the intron can typically bespliced out prior to translation by the host cell machinery. In the caseof the GALR2 cDNA, the location of the intron combined with clearconsensus sequences for 5′ and 3′ splice junctions (FIGS. 3A and 3B)confirm that the intervening sequence represents an intron. As shown inFIG. 3C, splicing of the intron at the indicated sites recreates an openreading frame within a highly conserved region of the GPCR family, atthe end of TMIII (LDR/Y). It is of interest to note that several GPCRshave previously been reported to contain introns at this location,including the human dopamine D3, D4, and D5 receptors, the rat substanceP receptor, and the human substance K receptor (Probst et al., 1992). Inparticular, the rat 5-HT₇ receptor (Shen et al., 1993) contains anintron in exactly the same location as is now reported for GALR2, withinthe AG/G codon for the highly conserved amino acid arginine at the endof TMIII (FIG. 3C).

[0316] To explore the possibility that incompletely or alternatelyspliced forms GALR2 mRNAs are present in the rat brain, RT-PCR usingGALR2 PCR primers that are located in the coding region but that spanthe location of the intron was carried out. The sequences of the PCRprimers are:

[0317] KS-1515 (Forward primer): 5′-CAAGGCTGTTCATTTCCTCATCTTTC (loopbetween TMs II and III)(SEQ.ID No. 12).

[0318] KS-1499 (Reverse primer): 5′-TTGGAGACCAGAGCGTAAACGATGG (end ofTMVII)(SEQ.ID No. 13).

[0319] The PCR products were separated by gel electrophoresis, blotted,and hybridized with a radiolabeled oligonucleotide probe representingthe predicted loop between TMs V and VI. The sequence of theoligonucleotide is:

[0320] KS-1540: 5′-AGTCGACCCGGTGACTGCAGGCTCAGGTTCCCAGCGCGCCAAACG (SEQ.ID No. 14).

[0321] RT-PCR analysis of GALR2 mRNA from various rat brain regions asdescribed above indicates the existence of PCR products that mayrepresent both the intronless (spliced) and intron-containing(incompletely spliced) forms of GALR2 (FIG. 5). In addition, PCRproducts intermediate in size between intronless and intron-containingproducts that hybridize at high stringency with the GALR2oligonucleotide probe KS-1540 are present and may represent additionalvariations in the GALR2 mRNA. One mechanism that could generate suchvariations is alternative splicing. These results suggest thatintronless transcripts exist in native tissue. A full-length intronlesscDNA encoding the rat GALR2 receptor has been amplified and subclonedfrom rat heart RNA, which when transiently or stably transfected intocells binds galanin with high affinity.

[0322] Northern Blot Analysis of GALR2 mRNA

[0323] To define the size and distribution of the mRNA encoding GALR2Northern blot analysis of poly A⁺ RNA from various rat tissues and brainregions was carried out. A ˜1.2 kb fragment of rat GALR2 containing theentire coding region but not containing the intron (FIG. 1) wasradiolabeled by random priming and used as a hybridization probe.Northern blots containing rat poly A⁺ RNA were hybridized at highstringency and apposed to film. A single transcript of ˜1.8-2.0 kb isdetected after a 4 day exposure of the autoradiogram at −80° C. usingKodak Biomax MS film with one Biomax MS intensifying screen. Within thebrain, the highest levels of GALR2 mRNA appear in hypothalamus (FIG.6A). Among various rat tissues, the GALR2 transcript is widely butunevenly distributed: GALR2 mRNA is observed in brain, lung, heart,spleen, and kidney, with lighter bands in skeletal muscle, liver, andtestis (FIG. 7A). Both Northern blots were reprobed with 1B15 to confirmthat similar amounts of mRNA were present in each lane (FIGS. 6B and7B).

[0324] Pharmacological Characterization of GALR2

[0325] The pharmacology of GALR2 was studied in COS-7 cells transientlytransfected with the GALR2 cDNA, K985. Membrane preparations of Cos-7cells transfected with K985 displayed specific binding to [¹²⁵I]porcinegalanin. Scatchard analysis of equilibrium saturation binding datayielded a K_(d)=150 pM with a B_(max)=250 fmol/mg protein. Thepharmacological properties of the protein encoded by the GALR2 cDNA wereprobed by measuring the binding affinities of a series of galaninanologs, and compared to those of the rat GALR1 receptor expressed inthe same host cell line. As shown in Table 1, both GALR1 and GALR2receptors showed a high affinity for galanin₍₁₋₂₉₎, the physiologicalligand of these receptors. Both receptors also displayed high affinityfor the truncated analogs galanin₍₁₋₁₆₎ and galanin₍₁₋₁₅₎. Furthermore,the binding of [¹²⁵I]porcine galanin to either GALR1 or GALR2 atconcentrations up to 100 μM was not displaced by porcine galanin₍₃₋₂₉₎.However, the GALR2 receptor has 540- and 4200-fold higher affinity for[D-Trp²]porcine galanin₍₁₋₂₉₎, and [D-Trp²]galanin₍₁₋₁₆₎, respectively,than the GALR1 subtype. Also, [Ala⁵]galanin₍₁₋₁₆₎ and[Phe²]galanin₍₁₋₁₅₎ were moderately selective, with 15- and 17-foldgreater affinities for the GALR2 receptor than for the GALR1 receptorsubtype, respectively. [Ala⁹]galanin₍₁₋₁₆₎ was the only analog that wasfound to have the opposite selectivity, with 70-fold higher affinity forthe GALR1 receptor than for the GALR2 receptor. Interestingly, these tworeceptor subtypes showed no significant differences in their bindingaffinities for the chimeric galanin antagonists, galantide, C7, M32,M35, and M40.

[0326] In LM(tk−) cells stably expressing the rat GALR2 receptor cDNA,porcine galanin₍₁₋₂₉₎ was found to inhibit the formation of cyclic AMPinduced by 10 μM forskolin. The effects of galanin were dose dependentwith an EC₅₀=0.26±0.13 nM (n=3) (FIG. 9A). In the same cell line porcinegalanin₍₁₋₂₉₎ stimulated the formation of [³H]inositol phosphates, withan EC₅₀=112 nM (FIG. 9B). The phosphoinositide response mediated by therat. GALR2 receptor suggests that this receptor can also couple to theintracellular calcium mobilization and diacylglycerol pathway. However,the 400-fold lower EC₅₀ of porcine galanin₍₁₋₂₉₎ suggests that the GALR2receptor couples with low efficiency to this signaling pathway. Insupport of this notion stands the observation that porcine galanin₍₁₋₂₉₎had no effect on intracellular calcium levels in COS-7 cells transfectedwith the cDNA encoding the rat GALR2 receptor. Thus, the data presentedherein suggest that the GALR2 receptor couples preferentially toG_(ialpha), since the stimulation of phosphoinositide metabolism andintracllular calcium mobilization are a hallmark or receptors to theG_(qα) family of G-proteins. Furthermore, the data presented herein alsoindicate that the inhibition of cAMP formation, as well as thestimulation of phosphoinositide metabolism, can be used as functionalassays to measure receptor activity in heterologous cell systemsexpressing the rat GALR2 receptor.

[0327] In subsequent experiments, the inhibitory effect of rat GALR2receptor stimulation on forskolin-stimulated cAMP accumulation inLM(tk−) cells could not be reproduced. However, the same LM(tk−) cellsyielded a reproducible PI hydrolysis response (Table 4), and inindependent binding assays a B_(max) of 4000 fmol/mg protein and a K_(d)of 1.1 nM when incubated with porcine ¹²⁵I-galanin. It is concluded thatin the cell lines studied thus far, the rat GalR2 is coupled primarilyto the activation of phospholipase C and subsequent inositol phosphatemetabolism, presumably through Gq or a related G protein. The PIresponse was evident as well in LM(tk−) cells stably transfected withthe rat GALR2 receptor cDNA lacking an intron in the coding region(L-rGALR2I-4, see Table 4); membranes from these cells were shown in anindependent experiment to bind porcine ¹²⁵I-galanin with a B_(max) of4800 fmol/mg membrane protein and a K_(d) of 0.2 nM.

[0328] The CHO cell line stably transfected with the rat GALR2 receptor(C-rGALR2-79) provided additional detail about the binding andsignalling properties of the receptor. Membranes from stably transfectedCHO cells were bound saturably by porcine ¹²⁵I-galanin with a B_(max) of520 fmol/mg membrane protein and a K_(d) of 0.53 nM. Peptides displacedthe porcine ¹²⁵I-galanin (Table 5) with binding affinities similar tothose generated from transiently transfected COS-7 cells (Table 1).Receptor stimulation resulted in phosphatidyl inositol hydrolysis buthad no effect on cAMP accumulation, again supporting the proposal thatthe rat GALR2 receptor is coupled primarily to phospholipase Cactivation through Gq or a related G protein. It was furtherdemonstrated that rat GALR2 receptor activation could be monitored byarachidonic acid release (Table 5). Of interest, it was observed thatthe EC₅₀ values from the PI hydrolysis assays were larger than the K_(i)values from binding assays whereas the EC₅₀ values from the arachidonicacid assays were comparable to the binding data. One possibilitysuggested by these data is that the calcium release induced by inositolphosphate metabolism leads to activation of phospholipase A2 andsubsequently to the hydrolysis of arachidonic acid from membranephospholipids. The lower EC₅₀ values in the arachidonic acid assays mayreflect an amplification process in the second messenger pathway, suchthat a maximal arachidonic acid response occurs at submaximal calciumconcentrations.

[0329] The stably transfected CHO cells were used to further explore thebinding and signalling properties of the rat GalR2 receptors (Table 6).The peptide binding profile was similar to that generated previouslywith transiently transfected COS-7 cells. Porcine, rat and human galaninbound with high affinity as did C-terminally truncated peptides as shortas galanin 1-12. Chimeric or putative “antagonist” peptides includingC7, galantide, M32, M35 and M40 displayed relatively high bindingaffinity except for C7 (Ki=47 nM). Galanin analogs containing D-Trp²(D-Trp²-galanin 1-29 and D-Trp²-galanin 1-16) retained measurablebinding affinity (K_(i)=41 and 110 nM, respectively). The N-terminallytruncated peptide galanin 3-29 was inactive.

[0330] Selected peptides were subsequently tested in the arachidonicacid release assays. Peptides with measurable EC₅₀ values mimicked themaximal effect of rat galanin (1 μM) on arachidonic acid release andwere classified as full agonists, including C7, galantide, M32, M35 andM40. The binding and functional profiles were in general agreement.Notable exceptions include D-Trp²-galanin 1-29, D-Trp²-galanin 1-16, andC7, all of which generated larger K_(i) values vs. EC₅₀ values; onepossibility is that these peptides were less stable in the binding assayvs. the functional assay. It is, therefore, concluded that thearachidonic acid release assay is useful for assessing peptide potencyand intrinsic activity for the rat GalR2 receptor when stably expressedin CHO cells.

[0331] Peptides were further evaluated for their ability to selectivelyactivate the rat GALR1 receptor (monitored in stably transfected LM(tk−)cells using the forskolin-stimulated cAMP accumulation assay) vs. therat GALR2 (monitored in stably transfected CHO cells using thearachidonic acid release assay). Data are reported in Table 7.D-Trp²-galanin was 8.5-fold less potent than galanin in the rat GALR2functional assay but >15000-fold less potent than galanin in the ratGALR1 functional assay. Similarly, D-Trp²-galanin 1-16 was 38-fold lesspotent than galanin in the rat GALR2 functional assay but >170,000-foldless potent than galanin in the rat GALR1 functional assay. It isconcluded that D-Trp²-galanin and analogous peptides may serve as usefultools with which to explore the function of GALR2 vs. GALR1 receptors innative tissues and physiological systems.

[0332] Feeding Assays

[0333] Rats were injected icv with either galanin, galanin derivatives,or saline. Cumulative food intake was measured over a period of 3 hours.Baseline food intake associated with the saline control was 1.5 gram. Amaximal food intake of 6.81 grams was observed after a 10 nmoleinjection of galanin. The ED50 for galanin is estimated to be 1 nmole.M40 was also tested in this paradigm. M40 was able to mimic the effectsof galanin, with a maximal food intake of 6.3 grams observed after a 50nmol injection. The ED50 for M40 is estimated to be 20 nmoles.

[0334] Heterologous Expression of GPCRs in Xenopus Oocytes

[0335] Heterologous expression of GPCRs in Xenopus oocytes has beenwidely used to determine the identity of signaling pathways activated byagonist stimulation (Gundersen et al., 1983; Takahashi et al., 1987).Application of porcine galanin (100-1000 nM) activates rapid inwardcurrents in 36 of 46 oocytes injected with 5-50 pg rGalR2 mRNA (FIG.13). Equimolar concentrations of C7 induces similar currents whereasgalanin 3-29 is inactive (0/11 oocytes). Oocytes injected with buffer(ND96) alone or 5-HT1a receptor mRNA do not exhibit detectable (<5 nA)responses to galanin (0/19). Current magnitudes in rGalR2 mRNA-injectedoocytes range from small fluctuations of less than 50 nA (excluded fromanalysis) to large rapid currents (up to 3 μA) resembling thoseactivated by stimulation of other receptors (alphala receptors—data notshown) that are known to couple to IP3 release and stimulation of Cl−current from the resulting increase in intracellular free Ca⁺⁺(Takahashi et al., 1987). The currents stimulated by galanin in oocytesexpressing rGalR2 are most likely mediated by the endogenouscalcium-activated Cl⁻ channel (Gunderson et al., 1983) because they areblocked in oocytes injected with 50 nl of 10 mM EGTA (5/5) and theydisplay a current-voltage relation that exhibits outward rectificationand a reversal potential of approximately −15 mV (data not shown).

[0336] Receptor Autoradiography

[0337] The relative proportion of the total [¹²⁵I]galanin bindingattributable to the GALR2 receptor was determined as the binding whichwas removed by 60 nM [D-Trp²]galanin₍₁₋₂₉₎. The numericalrepresentations in Table 2 indicate: 1) the relative intensity of thetotal binding obtained with [¹²⁵I]galanin, with +3 being the maximum;and 2) the relative amount of this binding attributable to GALR2, with+3 again being the maximum.

[0338] Total [¹²⁵I]galanin binding was observed in many regions of therat brain, and was especially intense in the forebrain, including theamygdala, parts of the hypothalamus and thalamus, the septum, and theventral hippocampus. Other regions with intense binding signals includedthe superior colliculus, the central gray, and the dorsal horn of thespinal cord. The inclusion of 5 μM porcine galanin in the incubationresulted in a complete displacement of [¹²⁵I]galanin binding from therat brain tissue sections. The use of 60 nM [D-Trp²]galanin₍₁₋₂₉₎partially displaced [¹²⁵I]galanin binding from many regions of the ratbrain.

[0339] The areas most affected by the GALR2 selective ligand were thelateral septum, the paraventricular hypothalamic nucleus, thecentromedial and centrolateral thalamic nuclei, the amygdalopiriformarea of the amygdala, and the superior colliculus. Other forebrainregions with lesser but still significant reductions in [¹²⁵I]galaninbinding included the piriform and entorhinal cortices, the globuspallidus, the supraoptic, lateral, and ventromedial hypothalamic nuclei,and the anterior, cortical, medial, and central amygdaloid nuclei. Inthe midbrain, pons and medulla, [D-Trp²]galanin₍₁₋₂₉₎ partially reducedthe total binding in the central gray, the raphe obscurus and raphemagnus, the parabrachial nucleus, the pontine reticular formation, thehypoglossal nucleus, and the gigantocellular reticular nucleus.

[0340] In contrast, there were a number of areas in which[D-Trp²]galanin₍₁₋₂₉₎ had little or no effect on the total [¹²⁵I]galaninbinding. Of these, the most striking were the nucleus of the lateralolfactory tract, the ventral hippocampus, and the dorsal horn of thespinal cord. Other areas in which significant binding remained includedthe olfactory bulb, the insular cortex, the islands of Calleja, thenucleus accumbens, the lateral habenula, the arcuate nucleus, and thespinal trigeminal nucleus.

[0341] Developmental in situ Hybridization

[0342] Using oligonucleotide probes, GalR2 mRNA appeared to bedevelopmentally regulated. At P1 and P5, film autoradiography of thehybridized brain sections revealed clear signals over many thalamicnuclei. In the hypothalamus, both the paraventricular and ventromedialnuclei were labeled. In addition, the superficial layers of neocortexcontained visible hybridization signal, as did the dorsal hippocampus.In the mesencephalon, a low level of hybridization signal was observedin the pretectal region.

[0343] Ribonuclease Protection Assay

[0344] RNA was isolated and assayed as described from: heart, striatedmuscle, liver, kidney, and CNS regions. CNS regions included: spinalcord, amygdala, hypothalamus, cerebral cortex, cerebellum, andhippocampus. The highest levels of rGalR2 were detected in thehypothalamus (FIG. 14). Lower amounts were found in heart, kidney,hippocampus amygdala, spinal cord, and cerebellum (FIG. 14). mRNA codingfor the rGalR2 was not detected in RNA extracted from striated muscle orliver.

[0345] Generation of Human GALR2 PCR Product

[0346] Using PCR primers designed against the fourth and sixthtransmembrane domains of the rat GALR2 sequence, NS 525 and NS526, a 300base pair fragment was amplified from 3 different lots of human genomicDNA. Sequence from all three human genomic DNAs were >98% identical anddisplayed 84% nucleotide identity to the rat GALR2 gene, between thesecond extracellular domain and the 5′ end of the sixth transmembrane.This level of homology is typical of a species homologue relationship inthe GPCR superfamily.

[0347] 5′ and 3′ RACE Analysis of Human GALR2

[0348] 5′ RACE was performed on human brain RNA to isolate hGALR2sequence upstream of the genomic PCR product above. Using nested reverseprimers from the fifth transmembrane domain of hGALR2, a 600 base pairfragment was amplified. The sequence of this RACE product displayed 91%nucleotide identity to rGALR2 from the 3′ end of the secondtransmembrane domain to the 5′ end of the fifth transmembrane domain.

[0349] 3′ RACE was performed on human lung RNA to determine the sequenceof the COOH terminus of hGALR2. Using nested forward primers from thefifth transmembrane domain of hGlR2, a 500 bp RACE product was generatedthat showed a 77% identity to nucleotides 1080-1139 of rGALR2. Thesequence of this RACE product downstream from this region showed lesshomology to rGALR2, and was presumed to represent the COOH terminus and3′ UT of the hGALR2 gene.

[0350] Construction and Screening of a Human Heart cDNA Library

[0351] To obtain a full-length hGALR2 clone, superpools of a human heartcDNA library were screened by PCR using primers BB153 and BB169. A 325base pair fragment was amplified from superpools 6, 9 and 16. Twopositive primary pools, 69 and 72, were identified from superpool 9, and1 positive primary pool, 121, was identified from superpool 16. Onepositive primary pool, 69, was subdivided into 48 pools of 3333individual clones and screened by PCR. Twelve positive subpools wereidentified and one, 69-11, was subdivided into 20 pools of 1200 clones,plated onto agar plates, and screened by southern analysis. Thirtycolonies that appeared positive were rescreened by PCR using primersBB167 and BB170, revealing 4 positive colonies. One of these, 69-11-5was chosen for further analysis. To evaluate whether this colonyrepresented a single clone, a dilution of the colony was amplified onagar plates and colonies were screened by PCR using primers BB167 andBB170. Five of 20 colonies were positive for hGALR2, indicating that69-11-5 was a mixture of 2 or more clones. One positive colony,69-11-5-3, designated BO29, was amplified as a single hGlR2 clone.Vector-anchored PCR revealed that BO29 is in the correct orientation forexpression, and encodes approximately 200 base pairs of 5′UT and 5000base pairs 3′UT. Preliminary single-stranded sequence analysis indicatesthat BO29 encodes an initiating methionine and a termination codon, andcontains an intron between the third and fourth transmembrane domainswhich is approximately 1.2 kb in length. 69-11-5 has been demonstratedto confer ¹²⁵I galanin binding in transfected COS-7 cells, as assessedby microscopic analysis of photoemulsion-dipped slides. In addition,COS-7 cells transfected with the single clone BO29 exhibit significantbinding of ¹²⁵I galanin in comparison with COS-7 cells transfected withcontrol vector. In preliminary radioligand binding experiments, ¹²⁵Iporcine galanin bound to membranes from COS-7 cells transfected withBO29, with a specific binding of 4900 fmol/mg, when the membranes (0.005mg/ml) were incubated with 0.4 nM porcine galanin for 30 min. at 30° C.No specific binding was detected to membranes from mock-transfectedCOS-7 cells when tested under the same conditions.

[0352] Human GALR2 Receptor Pharmacology

[0353] A human GALR2 receptor construct containing an intron in thecoding region of the cDNA (BO29) was prepared and transientlytransfected into COS-7 cells. Human GALR2 receptors expressed in theCOS-7 cell membranes were labeled by porcine ¹²⁵I-galanin with anapparent B_(max) of 4200 fmol/mg membrane protein and a K_(d) of 0.97nM. The peptide binding profile for the human GALR2 receptor (Table 8)resembled that reported previously for the rat GALR2 in COS-7 cellmembranes (Table 1).

[0354] A human GALR2 receptor cDNA construct lacking the intron in thecoding region was also prepared (BO39) and transiently transfected intoCOS-7 cells. In a preliminary experiment, membranes from transientlytransfected cells (membrane protein concentration=0.045 mg/ml) wereincubated with porcine ¹²⁵I-galanin (0.17 nM), and specific binding wasmeasured as 480 fmol/mg membrane protein. Assuming an estimated K_(d) of1 nM, the estimated B_(max) for this construct would be ˜3400 fmol/mgmembrane protein. Therefore, it is concluded that the absence of theintron in the coding region of the human GALR2 cDNA has no significanteffect on receptor expression or porcine ¹²⁵I-galanin binding.

[0355] Experimental Discussion

[0356] In order to clone additional members of the galanin receptorfamily, an expression cloning strategy based on the potential presenceof multiple galanin receptors in the hypothalamus was designed. Usingthis strategy a cDNA clone encoding a galanin receptor from rathypothalamus, termed GALR2, was isolated that is distinct from thepreviously cloned GALR1 receptors.

[0357] Transient transfection of the isolated cDNA (K985) encoding GALR2resulted in high affinity binding of [¹²⁵I]-porcine galanin. The highbinding affinity of the GALR2 receptor for galanin₍₁₋₂₉₎ and itstruncated analogs galanin₍₁₋₁₆₎ and galanin₍₁₋₁₅₎ strongly supports thenotion that the GALR2 receptor is a novel galanin receptor subtype. Boththe rat GALR1 and GALR2 receptors seem to bind preferentially to theamino terminus of galanin. Deletion of 13 or 14 amino acids from thecarboxyl terminus of galanin still yields peptides with high bindingaffinity at both the GALR1 and GALR2 receptors.

[0358] Furthermore, the truncation of the first two amino acids of theamino terminus led to a complete loss of affinity at both GALR1 andGALR2. Consistent with this notion are the findings that the chimericpeptides, which share identical amino acid sequences in the first 12amino acids with galanin had very similar binding affinities for eitherGAlR1 or GALR2 receptors. In spite of these similarities, thesubstitution of L-tryptophan with D-tryptophan in position 2 of porcinegalanin₍₁₋₂₉₎ ([D-Trp²]galanin₍₁₋₂₉₎) led to a 7,000-fold loss inaffinity at the GALR1 receptor compared to only a 14-fold reduction atthe GALR2 receptor. The same substitution in the truncated analoggalanin₍₁₋₁₆₎ led to a 4,200-fold reduction in affinity at the GAlR1receptor, and only a 6-fold reduction in affinity at the GALR2 receptor.These data suggest that galanin analogs, with modifications at the2-position, are better tolerated at the GALR2 receptor than at the GALR1receptor as long as the side chain is an aromatic moiety.

[0359] Conversely, the substitution of tyrosine with alanine in position9 of galanin₍₁₋₁₆₎, (i.e., to make [Ala]⁹ galanin) leads to a 680-foldreduction in affinity at the GALR1 receptor and to a 60,000-foldreduction in affinity at the GALR2 receptor. Altogether, the majordifferences in binding selectivity of the substituted analogs of galaninsuggest the existence of substantial differences in the binding domainsof these two receptor subtypes.

[0360] The existence of such structural differences between the GALR1and GALR2 receptors are indicative of the potential for the design anddiscovery of novel subtype selective compounds. In this regard, theexpression of the cDNA encoding the rat GALR2 receptors in cultured celllines provides a unique tool for the discovery of therapeutic agentstargeted at galanin receptors.

[0361] Localization of Galanin Receptors

[0362] The high affinity of [D-Trp²]galanin₍₁₋₂₉₎ for the cloned GALR2receptor (6 nM), and its low affinity for the GALR1 receptor (3 μM),makes it a useful tool for receptor autoradiographic studies. Thus,brain areas in which the total [¹²⁵I]galanin binding is significantlyreduced by [D-Trp²]galanin₍₁₋₂₉₎ are interpreted as areas containing ahigh proportion of GALR2 receptors, or other galanin receptors withsimilar high affinity for [D-Trp²]galanin₍₁₋₂₉₎. Those with lesserreductions are seen as regions containing a higher concentration ofGALR1 receptors. The lateral septum, the paraventricular hypothalamicnucleus, the centromedial and centrolateral thalamic nuclei, theamygdalopiriform area of the amygdala, and the superior colliculus allappear to contain primarily GALR2 receptors. In contrast, the nucleus ofthe lateral olfactory tract, the ventral hippocampus, and the dorsalhorn of the spinal cord appear to contain primarily GALR1 receptors. Thepredominance of the GALR1 receptor in these regions is consistent withpublished reports of the GALR1 messenger RNA localization (Parker etal., 1995; Gustafson et al., 1996). In most other regions, there appearsto be a significant overlap between the two subtypes.

[0363] While the functional implications of the GALR2 receptorlocalization are not well understood at present, there are a number ofphysiological processes attributable to galanin that could be mediatedby this receptor. These include feeding (paraventricular hypothalamicnucleus), cognition (septum and hippocampus), analgesia and/or sensoryprocessing (midline thalamic nuclei), and anxiety and depression(amygdala and hypothalamus).

[0364] The observation that galanin is co-released with norepinephrinefrom sympathetic nerve terminals suggests that galanin could act viagalanin receptors in the periphery to modulate nearly everyphysiological process controlled by sympathetic innervation. Additionaltherapeutic indications not directly related to localization (supra)include diabetes, hypertension, cardiovascular disorders, regulation ofgrowth hormone release, regulation of fertility, gastric ulcers,gastrointestinal motility/transit/absorption/secretion, glaucoma,inflammation, immune disorders, respiratory disorders (eg. asthma,emphysema).

[0365] The physiological and anatomical distribution ofgalanin-containing neurons suggests potential roles of galanin receptorsmediating effects on cognition, analgesia, neuroendocrine regulation,control of insulin release and control of feeding behavior. Ofparticular relevance to the role of the novel GALR2 receptor, are thosefunctions mediated by galanin receptors in the rat hypothalamus.

[0366] Studies in rats indicate that the injection of galanin in thehypothalamus increases food intake (Kyrouli et al, 1990, and Schick etal, 1993) and that this stimulatory effect of galanin is blocked byprior administration of M40 and C7 (Liebowitz and Kim, 1992; and Corwin,1993). The expression of the mRNA encoding the GALR1 receptor in the rathypothalamus, (Parker et al., 1995, Gustafson et al., 1996) and the factthat the novel GALR2 receptor was cloned from a cDNA library preparedfrom rat hypothalamus argues in favor of either receptor subtype to beinvolved in the regulation of feeding behavior (Parker et al., 1996).However, the evidence against the involvement of GALR1 in thestimulation of feeding behavior stems from the fact that M40 and C7 areknown to be agonists, and not antagonists, in cell lines expressing thecloned human and rat GALR1 receptors (Heuillet et al. 1994; Hale et al.1993; and Bartfai et al. 1993).

[0367] The distribution of GALR2 mRNA in the rat brain and periphery hasbeen determined by ribonuclease protection assay, in situ hybridization,Northen blot analysis and RT-PCR. The results of these studies suggestthat this receptor is potentially involved in mediating many of thephysiological roles ascribed to the peptide galanin. In the adult rat,localization of the GalR2 mRNA in the hypothalamus indicates a role forthis receptor in homeostatic mechanisms, including food intake andneuroendocrine regulation. The presence of GALR2 mRNA in the neocortexand dorsal hippocampus suggest an involvement in cognition, which isconsistent with documented changes in galanin and galanin receptorexpression during aging and in the brains of Alzheimer's patients(Chan-Palay, 1988; Leverenz et al., 1996). Galanin also hasantinociceptive effects, and the localization of GALR2 mRNA in thespinal cord (present investigation) and dorsal root ganglia (O'Donnellet al., 1996) implicate this receptor in pain neurotransmission. Thelocalization of GALR2 mRNA in the cerebellum is intriguing, as itsuggests a role for galanin and the GalR2 receptor in planned movementsand potentially in movement disorders.

[0368] In addition to the localization observed in adult animals, italso appears that the GALR2 mRNA is developmentally regulated, with thehighest levels observed early in postnatal development. Thus, it ispossible that this galanin receptor plays a role in. developmentalprocesses which occur during the first postnatal week, such as axonalguidance and synapse formation.

[0369] A unique pharmacological profile for the GALR2 receptor has beengenerated through binding and functional assays. This profile can beused to deduce the physiological function of the GalR2 receptor in vivo.Consider the agonist activity of galanin 1-16, for example. Galanin 1-16is reported to function as an agonist in various models of hypothalamic,pituitary and pancreatic function (Kask et al.) Galanin 1-16 is alsoreported to mimic the effects of galanin on the flexor reflex in therat. The N-terminally extended peptides galanin (−7) to (+) 29 andgalanin (−9) to (+) 29, also characterized as rat GALR2 agonists, canmimic the effects of galanin in a rat flexor reflex assay (Weisenfeld).Taken together, these data suggest a potential role for the rat GalR2receptor in a range of physiology or pathophysiology including diabetes,pain, reproduction, obesity and eating disorders.

[0370] The agonist activity of M40 in GALR2 in vitro assays isparticularly intriguing when viewed in the context of behavioral feedingmodels. From the literature, one might conclude that the agonistactivity of M40 in vitro is in apparent conflict with the antagonistactivity reported for M40 in behavioral models of food intake, and thatthe GALR2 receptor is therefore unlikely to mediate the feedingresponse. The data generated and reported in the subject application donot support this conclusion. Rather, the data from behavioral feedingmodels indicate that M40 is an orexigenic peptide whose maximal effectis comparable to that for galanin itself. The agonist activity reportedherein for M40 both in vitro and in vivo is consistent with the proposalthat the rat GALR2 receptor mediates the stimulatory effect of galaninon food intake in the central nervous system. These data further suggestthat the rat GALR2 receptor represents a target for the design oftherapeutic compounds for the treatment of obesity and relateddisorders. TABLE 1 Binding of galanin peptide analogs to the recombinantrat GALR1 and GALR2 receptors transiently expressed in COS 7 cells.GALR1 (pKi) GALR2 (pKi) Analog Mean SEM* Mean SEM porcine galanin₍₁₋₂₉₎9.34 0.15 9.35 0.14 [D-Trp²]porcine 5.46 0.04 8.19 0.26 galanin₍₁₋₂₉₎[Phe²]porcine 5.99 0.13 5.64 0.11 galanin₍₁₋₂₉₎ [D-Ala⁷]porcine 8.660.04 8.76 0.09 galanin₍₁₋₂₉₎ galanin₍₁₋₁₆₎ 8.66 0.01 8.76 0.13[D-Trp²]galanin₍₁₋₁₆₎ 4.40 0.09 8.02 0.10 [Ala⁵]galanin₍₁₋₁₆₎ 6.27 0.057.46 0.13 [Ala⁹]galanin₍₁₋₁₆₎ 5.83 0.02 3.98 0.10 galanin₍₁₋₁₅₎ 8.470.04 9.19 0.06 [Phe²]galanin₍₁₋₁₅₎ 4.63 0.03 5.85 0.49 porcinegalanin₍₃₋₂₉₎ <4.0 <4.0 galantide 8.02 0.08 8.70 0.07 C-7 7.79 0.01 7.720.09 M32 9.21 0.10 9.23 0.05 M35 9.48 0.07 9.24 0.10 M40 8.44 0.09 9.140.21

[0371] TABLE 2 Distribution of [¹²³I]galanin binding in rat brain. Totalbinding is compared to the amount attributable to GALR2 (as indicated bydisplacement of [¹²⁵I]galanin by 60 nM [D-Trp²]porcine galanin₍₁₋₂₉₎).Total Putative [¹²⁵I]Gal GALR2 Potential Region binding sitesApplications Olfactory bulb +3 +1 Modulation of olfactory sensationAnterior olfactory n. +3 +1 Modulation of olfactory sensation Cortexdorsal neocortex, +1 +1 Sensory layer 4 integration piriform +2 +1Modulation of olfactory sensation agranular insular +3 +1 Processing ofvisceral information entorhinal +2 +1 dorsal endopiriform +2 +1Claustruin +2 +1 Visual processing Basal ganglia n. accumbens +2 0Modulation of olfactory tubercle +2 +1 dopaminergic globus pallidus +1+1 function islands of Calleja +3 +1 Septal area Cognitive lateralseptum +3 +2 enhancement via diagonal band n. +2 0 cholinergic systemHypothalamus anterior +1 0 Neuroendocrine supraoptic n. +2 +1 regulationparaventricular +2 +2 Appetite/obesity ventromedial +2 +1 arcuate +1 0lateral +2 +1 medial mammillary +2 +1 Thalamus Analgesia/sensoryparaventricular n. +1 0 modulation centromedial +3 +2 paracentral +3 +1rhomboid +1 0 reuniens +2 +1 mediodorsal +2 0 reticular n. +1 +½centrolateral n. +3 +2 zona incerta +2 +1 lateral dorsal +1 +½ habenula+3 +1 Anxiety/sleep disorders Hippocampus Cognition Cal, ventral +3 0enhancement/ subiculum +2 +1 ischaemia Amygdala Anxiolytic, bed n. stria+3 +1 appetite, terminalis depression n. lateral olfactory +3 0 tractAmygdala Anxiolytic, anterior +2 +1 appetite, medial +3 +1 depressioncortical +2 +1 central +3 +1 amygdalohippocampal +2 0 amygdalopiriform+3 +2 Midbrain superior colliculus +3 +2 Visual function raphe obscurus+2 +1 Analgesia central gray +2 +1 Analgesia Pons/medulla raphe magnus+2 +1 Analgesia parabrachial n. +2 +1 pontine ret. n. +2 +1reticulotegmental +2 +1 gigantocellular +2 +1 motor trigeminal +1 0spinal trigeminal +3 +1 Migraine hypoglossal n. +2 +1 Motor coordinationarea postrema +1 0 Spinal cord dorsal horn +3 +1 Analgesia

[0372] TABLE 3 Northern blot hybridization of GALR2 receptor in brainand various peripheral rat tissues. Mean Tissue Blot 1 Blot 2 SignalTherapeutic Indications Heart +++ ++ 2.5 Cardiovascular Indications(including hypertension and heart failure) Brain ++++ ++++ 4.0obesity/feeding, analgesia, cognition enhancement, Alzheimer's disease,depression, anxiety, sleep disorders, Parkinson's disease, traumaticbrain injury, convulsion/epilepsy Spleen ++ ++ 2.0 Immune functions,hematopoiesis Lung ++++ ++++ 4.0 Respiratory disorders, asthma,emphysema, lung cancer diagnostics Liver ++ − 1.0 Diabetes Skeletal + ++1.5 Diabetes Muscle Kidney +++ +++ 3.0 Hypertension, electrolytebalance, diuretic, anti-diuretic Testis +++ + 2.0 Reproductive function

[0373] TABLE 4 Inositol phosphate hydrolysis in LM(tk-) cells stablytransfected with GALR2. EC₅₀ PI(nM) EC₅₀ PI (nM) rat GALR2 L-rGALR2I-4Peptide (with intron) (intronless) porcine galanin 21 14 M35 29 28D-Trp²-galanin 1-16 1380 660 D-Trp²-galanin 1-29 200 230 galanin 1-16 6518 M40 28 47 M32 13 35

[0374] TABLE 5 Rat GALR2 receptors stably transfected in CHO Comparisonof binding data, phosphatidyl inositol release, and arachidonic acidrelease in C-rGalR2-79. K_(i) from EC₅₀ from EC₅₀ from porcine PIarachi- ¹²⁵I-galanin hydrolysis donic acid binding assays assays Peptideassays (nM) (nM) (nM) rat galanin 0.52 14 0.67 porcine 0.94 15 1.3galanin porcine 3.5 91 2.6 galanin 1-16 D-Trp²-galanin 110 590 50 1-16

[0375] TABLE 6 CHO GALR2 pharmacology: binding (K_(i) vs. ¹²⁵I- porcinegalanin) vs. function (arachidonic acid hydrolysis) Rat GALR2 Rat GALR2K_(i), EC₅₀ AA, C-rGalR2- C-rGalR2-79 Peptide 79 (nM) (nM) Human galanin1.2 Porcine galanin 0.94 1.3 rat galanin 0.52 0.67 porcine gal −7 to +293.0 porcine galanin −9 to +29 4.0 porcine galanin 3-iodo-L- 0.8Tyr9-galnin porcine galanin 3-iodo-L- 1.0 Tyr26 galanin porcinePhe2-galanin >1000 porcine D-Trp2-galanin 41 11 D-Trp2-3-iodo-L-Tyr9-3.0 galanin porcine D-Trp2-3-iodo-L- 6.0 Tyr26-galanin D-Ala7-galanin6.2 5.4 porcine galanin 3-29 >1000 >1000 porcine galanin9-29 >1000 >1000 porcine galanin 17-29 >1000 porcine galanin 1-16 3.52.6 porcine Ala2-galanin 1-16 >1000 porcine D-Trp2-galanin 1-16 110 50porcine Ala5-galanin 1-16 >620 porcine Ala9-galanin 1-16 >1000 porcinegalanin 1-15 1.5 2.3 Phe2-galanin 1-16 >1000 >1000 porcine galanin 1-122.1 2.3 porcine galanin 1-9 >1000 >1000 C7 48 2.4 Galantide 4.9 0.93 M323.4 2.5 M35 5.8 1.3 M40 3.5 2.7

[0376] TABLE 7 Peptide-dependent activation of rat GALR1 vs. rat GALR2.LM (tk-) C-RGalR2-79 Rat GALR1 arachidonic acid cAMP assay assay PeptideEC₅₀ (nM) EC₅₀ (nM) Porcine galanin 0.06 1.3 rat galanin 0.05 0.67porcine D-Trp²-galanin >850 11 porcine galanin 3-29 >1000 >1000 porcinegalanin 1-16 0.34 2.6 porcine >1000 50 D-Trp²-galanin 1-16 C7 0.52 2.4Galantide 0.08 0.93 M32 0.34 2.5 M35 0.15 1.3 M40 0.82 2.7

[0377] TABLE 8 Peptide binding profile: Human GALR2 vs. rat GALR2transiently expressed in COS-7 Human GALR2 K_(i) Rat GALR2 K_(i) Peptide(nM) (nM) porcine galanin 1-16 15 7.2* porcine galanin 0.72 0.45 M40 5.30.72 porcine D-Trp²- 290 52* galanin M32 7.9 12* rat galanin 1.0 0.52*

[0378] References

[0379] Ahrén, B. and S. Lindskog (1992) Int. J. Pancreatol. 11:147-160.

[0380] Amiranoff, B. A. M. Lorinet, and M. Laburthe (1991) Eur. J.Biochem. 195:459-463.

[0381] Amiranoff, B. A. L. Servin, C. Rouyer-Fessard, A. Couvineau, K.Tatemoto, and M. Laburthe (1987) Endocrin. 121:284-289.

[0382] Aruffo, A. and B. Seed (1987) Proc. Natl. Acad. Sci. USA84:8573-8577.

[0383] Bhathena, S. J., H. K. Oie, A. F. Gazdar, N. R. Voyles, S. D.Wilkins, and L. Recant (1982) Diabetes 31:521-531.

[0384] Bartfai,T., K. Bedecs, T. Land, Ü. Langel, R. Bertorelli, P.Girotti, S. Consolo, Y.-J. Yu, Z. Weisenfeld-Hallin, S. Nilsson, V.Pieribone, and T. Hökfelt (1991) Proc. Natl. Acad. Sci. USA88:10961-10965.

[0385] Bartfai,T., T. Hokfelt, and U. Langel, Crit. Rev. Neurobiol.7:229-274.

[0386] Bartfai,T., Ü. Langel, K. Bedecs, S. Andell, T. Land, S.Gregersen, B. Ahren, P. Girotti, S. Consolo, R. Corwin, J. Crawley, X.Xu, Z. Weisenfeld-Hallin, and T. Hökfelt (1993) Proc. Natl. Acad. Sci.USA 88:11287-11291.

[0387] Bennet, W. M., S. F. Hill, M. A. Ghatei, and S. R. Bloom (1991)J. Endocrin. 130:463-467.

[0388] Borden, L. A., K. E. Smith., P. R. Hartig, T. A. Branchek, and R.L. Weinshank (1992) J. Biol. Chem. 267: 21098-21104.

[0389] Borden, L. A., K. E. Smith, E. L. Gustafson, T. A. Branchek, andR. L. Weinshank (1995) J. Neurochem. 64977-984.

[0390] Boyle, M. R., C. B. Verchere, G. McKnight, S. Mathews, K. Walker,and G. J. Taborsky, Jr. (1994) Reg. Peptides 50:1-11.

[0391] Bradford, M. M. (1976). A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal. Biochem. 72: 248-254.

[0392] Burbach, J. P. and O. C. Meijer (1992) Eur. J. Pharmacol.227:1-18.

[0393] Burgevin, M.-C., Loquet, I., Quarteronet, D., and Habert-Ortoli,E. (1995) J. Molec. Neurosci., 6:33-41.

[0394] Burns, C. M., Chu, H., Rueter, S. M., Sanders-Bush, E., and R. B.Erneson. (1996) Neuroscience Abstracts 385.9.

[0395] Bush, A. W., Borden, L. A., Greene, L. A., and Maxfield, F. R.(1991) J. Neurochem. 57:562-574.

[0396] Chan-Palay, V. (1988) J.Comp.Neurol. 273:543-557. Chen, Y., A.Fournier, A. Couvineau, M. Laburthe, and B. Amiranoff (1993) Proc. Natl.Acad. Sci. USA 90:3845-3849.

[0397] Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J.Rutter. (1979) Biochemistry 18:5294-5299.

[0398] Chu, H., Burns, C., Canton, H., Emeson, R. B., and E.Sanders-Bush. (1996) Neuroscience Abstracts 385.10.

[0399] Consolo, S., R. Bertorelli, P. Girotti, C. La Porta, T. Bartfai,M. Parenti, and M. Zambelli (1991) Neurosci. Lett. 126:29-32.

[0400] Crawley, J. N. (1993) Behav. Brain Res. 57:133-141.

[0401] Crawley, J. N., J. K. Robinson, Ü. Langel, and T. Bartfai (1993)Brain. Res. 600:268-272.

[0402] Cullen, B. (1987). Use of eurkaryotic expression technology inthe functional analysis of cloned genes. Methods Enzymol. 152: 685-704.

[0403] D'Andrea, A. D., H. F. Lodish, and G. W. Gordon (1989) Cell57:277-285.

[0404] Fisone, G., C. F. Wu, S. Consolo, Ö. Nördstrom, N. Brynne, T.Bartfai, T. Melander, T. Hökfelt (1987) Proc. Natl. Acad. Sci USA84:7339.

[0405] Gearing, D. P., King, J. A.,Gough, N. M. and Nicola N. A. (1989)EMBO J. 8:3667-3676.

[0406] Gerald, C., M. Walker, T. Branchek, and R. Weinshank (1994) DNAEncoding a Human Neuropeptide Y/Peptide YY (Y2) Receptor and UsesThereof, U.S. patent application U.S. Ser. No. 08/192,288, filed Feb. 3,1994.

[0407] Gillison, S. L., and W. G. Sharp (1994) Diabetes 43:24-32.Gregersen, S., S. Lindskog, T. Land, U. Langel, T. Bartfai, and B. Ahren(1993) Eur J. Pharmacol. 232:35-39.

[0408] Gu, Z.-F., W. J. Rossowski, D. H. Coy, T. K. Pradhan, and R. T.Jensen (1993) J. Phamacol. Exper. Ther. 266:912-918.

[0409] Gu, Z.-F., Pradhan, T. K., Coy, D. H., and Jensen, R. T. (1995)J. Pharmacol. Exp. Ther. 272:371-378.

[0410] Gubler, U abd B. J. Hoffman. (1983). A simple and very efficientmethod for generating cDNA libraries. Gene. 25, 263-269

[0411] Gundersen, C. B., R. Miledi, and I. Parker. 1983. Proc. R. Soc.London Ser. B 219:103-109.

[0412] Gustafson, E. L., Smith, K. E., Durkin, M. M., Gerald, C., andBranchek, T. A. (1996) Neuroreport, 7:953-957.

[0413] Habert-Ortoli, E., Amiranoff, B., Loquet, I., Laburthe, M., andJ.-F. Mayaux (1994) Proc. Natl. Acad. Sci.USA 91:9780-9783.

[0414] Hedlund, P. B., N. Yanaihara, and K. Fuxe (1992) Eur. J. Pharm.224:203-205.

[0415] Heuillet, E., Bouaiche, Z., Menager, J., Dugay, P., Munoz, N.,Dubois, H., Amiranoff, B., Crespo, A., Lavayre, J., Blanchard, J.-C.,and Doble, A. (1994) Eur. J. Pharmacol., 269:139-147.

[0416] Kaplan, L. M., S. M. Gabriel, J. I. Koenig, M. E. Sunday, E. R.Spindel, J. B. Martin, and W. W. Chin (1988) Proc. Natl. Acad. Sci.USA85:7408-7412.

[0417] Kieffer, B., Befort, K., Gaveriaux-Ruff, C. and Hirth, C. G.(1992). The δ-opioid receptor: Isolation of a cDNA by expression cloningand pharmacological characterization. Proc. Natl. Acad. Sci. USA89:12048-12052.

[0418] Kluxen, F. W., Bruns, C. and Lubbert H. (1992). Expressioncloning of a rat brain somatostatin receptor cDNA. Proc. Natl. Acad.Sci. USA 89:4618-4622.

[0419] Kornfeld, R. and Kornfeld, S. (1985). Assembly of asparaginelinked oligosaccharides. Annu. Rev. Biochem. 54:631-664.

[0420] Kozak, M. (1989). The scanning model for translation: an update.J. Cell Biol. 108: 229-241.

[0421] Kozak, M. (1991). Structural features in eukaryotic mRNAs thatmodulate the initiation of translation. J. Biol. Chem. 266: 19867-19870.

[0422] Kyrkouli, S. E., B. G. Stanley, R. D. Seirafi and S. F. Leibowitz(1990) Peptides 11:995-1001.

[0423] Lagny-Pourmir, I., A. M. Lorinet, N. Yanaihara, and M. Laburthe(1989) Peptides 10:757-761.

[0424] Landschultz, W. H., Johnson, P. F. and S. L. McKnight. (1988).The leucine zipper: a hypothetical structure common to a new class ofDNA binding proteins. Science 240: 1759-1764.

[0425] Leibowitz, S. F. and T. Kim (1992) Brain Res. 599:148-152.

[0426] Maggio, R., Vogel Z. and J. Wess. (1993). Coexpression studieswith mutant muscarinic/adrenergic receptors provide evidence forintermolecular “cross-talk” between G-protein-linked receptors. Proc.Natl. Acad. Sci. USA 90: 3103-3107.

[0427] McCormick, M. (1987). Sib Selection. Methods in Enzymology, 151:445-449.

[0428] Melander, T., C. Köhler, S. Nilsson, T. Hökfelt, E. Brodin, E.Theodorsson, and T. Bartfai (1988) J. Chem. Neuroanat. 1:213-233.

[0429] Merchenthaler, I., F. J. López, and A. Negro-Vilar (1993) Prog.Neurobiol. 40:711-769.

[0430] Miller, J. and Germain, R. N. (1986). Efficient cell surfaceexpression of class II MHC molecules in the absence of associatedinvariant chain. J. Exp. Med. 164: 1478-1489.

[0431] Ögren, S.-O., T. Hökfelt, K. Kask, Ü. Langel, and T. Bartfai(1992) Neurosci. 51:1.

[0432] Palazzi, E., G. Fisone, T. Hökfelt, T. Bartfai, and S. Consolo(1988) Eur. J. Pharmacol. 148:479.

[0433] Parker, E. M., Izzarelli, D., Nowak, H., Mahle, C., Iben, L.,Wang, J., and Goldstein, M. E. (1995) Mol. Brain Res., 34:179-189.

[0434] Post, C., L. Alari, and T. Hökfelt (1988) Acta Physiol. Scand.132:583.

[0435] Probst, W. C., Snyder, L.-A., Schuster, D. I., Brosius, J andSealfon, S. C. (1992). Sequence alignment of the G-protein coupledreceptor superfamily. DNA and Cell Bio. 11: 1-20.

[0436] Quick, M. W. and H. A. Lester. 1994. Methods for expression ofexcitability proteins in Xenopus oocytes. Meth. Neurosci. 19:261-279.

[0437] Sanger, S. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467.

[0438] Servin, A. L., B. Amiranoff, C. Rouyer-Fessard, K. Tatemoto, andM. Laburthe (1987) Biochem. Biophys. Res. Comm. 144:298-306.

[0439] Shen, Y., Monsma, F. J. Jr., Metcalf, M. A., Jose, P. A.,

[0440] Hamblin, M. W., and Sibley, D. R. (1993) Molecular Cloning andExpression of a 5-Hydroxytryptamine₇ Serotonin Receptor Subtype. J.Biol. Chem. 268:18200-18204.

[0441] Sims, J. E., C. J. March, D. Cosman, M. B. Widmer, H. R.Macdonald, C. J. McMahan, C. E. Grubin, J. M. Wignal, J. L. Jackson, S.M. Call, D. Freind, A. R. Alpert, S. Gillis, D. L. Urdal, and S. K.Dower (1988) Science 241:585-588.

[0442] Skofitsch, G. and D. M. Jacobowitz (1985) Peptides 6:509-546.

[0443] Skofitsch, G., M. A. Sills, and D. M. Jacobowitz (1986) Peptides7:1029-1042.

[0444] Smith, K. E., L. A. Borden, P. R. Hartig, T. Branchek, and R. L.Weinshank (1992) Neuron 8: 927-935.

[0445] Smith, K. E., L. A. Borden, C-H. D. Wang, P. R. Hartig, T. A.Branchek, and R. L. Weinshank (1992a) Mol. Pharmacol. 42:563-569.

[0446] Smith, K. E., S. G. Fried, M. M. Durkin, E. L. Gustafson, L. A.Borden, T. A. Branchek, and R. L. Weinshank (1995) FEBS Letters,357:86-92.

[0447] Sundström, E., T. Archer, T. Melander, and T. Hökfelt (1988)Neurosci. Lett. 88:331.

[0448] Takahashi, T., E. Neher, and B. Sakmann. 1987. Rat brainserotonin receptors in Xenopus oocytes are coupled by intracellularcalcium to endogenous channels. Proc. Natl. Acad. Sci. USA 84:5063-6067.

[0449] Tempel, D. L., K. J. Leibowitz, and S. F. Leibowitz (1988)Peptides 9:300-314.

[0450] Vrontakis, M. E., L. M. Peden, M. L Duckworth, and H. G. Friesen(1987) J. Biol. Chem. 262:16755-16760.

[0451] Warden, D. and H. V. Thorne. (1968). Infectivity of polyoma virusDNA for mouse embryo cells in presence of diethylaminoethyl-dextran. J.Gen. Virol. 3: 371.

[0452] Wiesenfeld-Hallin, Z., X. J. Xu, J. X. Hao, and T. Hökfelt (1993)Acta Physiol.Scand 147:457-458.

[0453] Wiesenfeld-Hallin, Z., et al. (1992) Proc. Natl. Acad. Sci. USA89:3334-3337.

[0454] Wynick D., D. M. Smith, M. Ghatei, K. Akinsanya, R. Bhogal, P.Purkiss, P. Byfield, N. Yanaihara, and S. R. Bloom (1993) Proc. Natl.Acad. Sci. USA 90:4231-4245.0

1 30 1 1193 DNA Rattus norvegicus 1 caagacccgg acagctgcgg gagcggcgtccactttggtg ataccatgaa tggctccggc 60 agccagggcg cggagaacac gagccaggaaggcggtagcg gcggctggca gcctgaggcg 120 gtccttgtac ccctattttt cgcgctcatcttcctcgtgg gcaccgtggg caacgcgctg 180 gtgctggcgg tgctgctgcg cggcggccaggcggtcagca ccaccaacct gttcatcctc 240 aacctgggcg tggccgacct gtgtttcatcctgtgctgcg tgcctttcca ggccaccatc 300 tacaccctgg acgactgggt gttcggctcgctgctctgca aggctgttca tttcctcatc 360 tttctcacta tgcacgccag cagcttcacgctggccgccg tctccctgga caggtatctg 420 gccatccgct acccgctgca ctcccgagagttgcgcacac ctcgaaacgc gctggccgcc 480 atcgggctca tctgggggct agcactgctcttctccgggc cctacctgag ctactaccgt 540 cagtcgcagc tggccaacct gacagtatgccacccagcat ggagcgcacc tcgacgtcga 600 gccatggacc tctgcacctt cgtctttagctacctgctgc cagtgctagt cctcagtctg 660 acctatgcgc gtaccctgcg ctacctctggcgcacagtcg acccggtgac tgcaggctca 720 ggttcccagc gcgccaaacg caaggtgacacggatgatca tcatcgtggc ggtgcttttc 780 tgcctctgtt ggatgcccca ccacgcgcttatcctctgcg tgtggtttgg tcgcttcccg 840 ctcacgcgtg ccacttacgc gttgcgcatcctttcacacc tagtttccta tgccaactcc 900 tgtgtcaacc ccatcgttta cgctctggtctccaagcatt tccgtaaagg tttccgcaaa 960 atctgcgcgg gcctgctgcg ccctgccccgaggcgagctt cgggccgagt gagcatcctg 1020 gcgcctggga accatagtgg cagcatgctggaacaggaat ccacagacct gacacaggtg 1080 agcgaggcag ccgggcccct tgtcccaccacccgcacttc ccaactgcac agcctcgagt 1140 agaaccctgg atccggcttg ttaaaggaccaaagggcatc taacagcttc tag 1193 2 372 PRT Rattus norvegicus 2 Met Asn GlySer Gly Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu Gly 1 5 10 15 Gly SerGly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe 20 25 30 Ala LeuIle Phe Leu Val Gly Thr Val Gly Asn Ala Leu Val Leu Ala 35 40 45 Val LeuLeu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile 50 55 60 Leu AsnLeu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro 65 70 75 80 PheGln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu 85 90 95 LeuCys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser 100 105 110Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg 115 120125 Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala 130135 140 Ala Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr145 150 155 160 Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr ValCys His 165 170 175 Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp LeuCys Thr Phe 180 185 190 Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu SerLeu Thr Tyr Ala 195 200 205 Arg Thr Leu Arg Tyr Leu Trp Arg Thr Val AspPro Val Thr Ala Gly 210 215 220 Ser Gly Ser Gln Arg Ala Lys Arg Lys ValThr Arg Met Ile Ile Ile 225 230 235 240 Val Ala Val Leu Phe Cys Leu CysTrp Met Pro His His Ala Leu Ile 245 250 255 Leu Cys Val Trp Phe Gly ArgPhe Pro Leu Thr Arg Ala Thr Tyr Ala 260 265 270 Leu Arg Ile Leu Ser HisLeu Val Ser Tyr Ala Asn Ser Cys Val Asn 275 280 285 Pro Ile Val Tyr AlaLeu Val Ser Lys His Phe Arg Lys Gly Phe Arg 290 295 300 Lys Ile Cys AlaGly Leu Leu Arg Pro Ala Pro Arg Arg Ala Ser Gly 305 310 315 320 Arg ValSer Ile Leu Ala Pro Gly Asn His Ser Gly Ser Met Leu Glu 325 330 335 GlnGlu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu 340 345 350Val Pro Pro Pro Ala Leu Pro Asn Cys Thr Ala Ser Ser Arg Thr Leu 355 360365 Asp Pro Ala Cys 370 3 2200 DNA Rattus norvegicus 3 caagacccggacagctgcgg gagcggcgtc cactttggtg ataccatgaa tggctccggc 60 agccagggcgcggagaacac gagccaggaa ggcggtagcg gcggctggca gcctgaggcg 120 gtccttgtacccctattttt cgcgctcatc ttcctcgtgg gcaccgtggg caacgcgctg 180 gtgctggcggtgctgctgcg cggcggccag gcggtcagca ccaccaacct gttcatcctc 240 aacctgggcgtggccgacct gtgtttcatc ctgtgctgcg tgcctttcca ggccaccatc 300 tacaccctggacgactgggt gttcggctcg ctgctctgca aggctgttca tttcctcatc 360 tttctcactatgcacgccag cagcttcacg ctggccgccg tctccctgga caggtgagtg 420 aacatcggagaactattgta tctgagatag gggcttgggc tggagtcact acacagggga 480 tccagaaggcatgagcagaa tgggcgagaa cactgaaatt acaaagtggc ctgaggccgt 540 gaaacgcaagggggagggag attaagactc agtgactgag agtgtctaag tcgatgggag 600 aaatcgggtctctggggtcc tcgcattatt actgcttgag ttaaatgtct ctgtgaaaca 660 ttgcagttctcaggccagag ttggcaggaa aagtaactcg ccagtgttca gatgctgttt 720 gagagctgcagagaagcatc tgcttcttag caccaagctc agcacctggg gcgttgtccg 780 gcgccttaggcttaggactg ggctgtgctg tgttaagacc catgctcaag tccaacggag 840 tgtaagcgagggctcctagc tgacacccag agccctccag gccaaggctc ccctcaccga 900 gatgccagcggttttatgct ccttccatag gtaaaggacc cagaaagaaa catccagtat 960 gcccggagggatcttgactg gaaaagactg aatcctggtc tggtgacctt agttccctgc 1020 cctttcacatcacttggaca ttcccacaga agagcggtga agaggcggtg gtccttattc 1080 tcctctggtttccactgagt gcaacatgtg cgtcctgagt acgctggagg gactcacaaa 1140 atttcagctttctttaggag tttccttgct gtagtttgac ccaagtcttc tccaggtttc 1200 tgtcagaacctcaggcatga gggatctgcc tcccctggtt gtcaccagag gataacaatc 1260 actgcccccagaaatccaga cagattctac aacttttagt cttcggtgtt ttgggggtgc 1320 cccttcacgtggagtaggtc ggtggccaca ttcccaggag tgacaatagc ctagcagtga 1380 atcctctcgcttagctgatg cccccccact gtccccacag gtatctggcc atccgctacc 1440 cgctgcactcccgagagttg cgcacacctc gaaacgcgct ggccgccatc gggctcatct 1500 gggggctagcactgctcttc tccgggccct acctgagcta ctaccgtcag tcgcagctgg 1560 ccaacctgacagtatgccac ccagcatgga gcgcacctcg acgtcgagcc atggacctct 1620 gcaccttcgtctttagctac ctgctgccag tgctagtcct cagtctgacc tatgcgcgta 1680 ccctgcgctacctctggcgc acagtcgacc cggtgactgc aggctcaggt tcccagcgcg 1740 ccaaacgcaaggtgacacgg atgatcatca tcgtggcggt gcttttctgc ctctgttgga 1800 tgccccaccacgcgcttatc ctctgcgtgt ggtttggtcg cttcccgctc acgcgtgcca 1860 cttacgcgttgcgcatcctt tcacacctag tttcctatgc caactcctgt gtcaacccca 1920 tcgtttacgctctggtctcc aagcatttcc gtaaaggttt ccgcaaaatc tgcgcgggcc 1980 tgctgcgccctgccccgagg cgagcttcgg gccgagtgag catcctggcg cctgggaacc 2040 atagtggcagcatgctggaa caggaatcca cagacctgac acaggtgagc gaggcagccg 2100 ggccccttgtcccaccaccc gcacttccca actgcacagc ctcgagtaga accctggatc 2160 cggcttgttaaaggaccaaa gggcatctaa cagcttctag 2200 4 1365 DNA Homo sapiens CDS(102)..(1265) 4 agtcgcacta ggagttgcag cggccgcagc cccgggagct tcccgctcgcggagacccag 60 acggctgcag gagcccgggc agcctcgggg tcagcggcac c atg aac gtctcg ggc 116 Met Asn Val Ser Gly 1 5 tgc cca ggg gcc ggg aac gcg agc caggcg ggc ggc ggg gga ggc tgg 164 Cys Pro Gly Ala Gly Asn Ala Ser Gln AlaGly Gly Gly Gly Gly Trp 10 15 20 cac ccc gag gcg gtc atc gtg ccc ctg ctcttc gcg ctc atc ttc ctc 212 His Pro Glu Ala Val Ile Val Pro Leu Leu PheAla Leu Ile Phe Leu 25 30 35 gtg ggc acc gtg ggc aac acg ctg gtg ctg gcggtg ctg ctg cgc ggc 260 Val Gly Thr Val Gly Asn Thr Leu Val Leu Ala ValLeu Leu Arg Gly 40 45 50 ggc cag gcg gtc agc act acc aac ctg ttc atc cttaac ctg ggc gtg 308 Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile Leu AsnLeu Gly Val 55 60 65 gcc gac ctg tgt ttc atc ctg tgc tgc gtg ccc ttc caggcc acc atc 356 Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln AlaThr Ile 70 75 80 85 tac acc ctg gac ggc tgg gtg ttc ggc tcg ctg ctg tgcaag gcg gtg 404 Tyr Thr Leu Asp Gly Trp Val Phe Gly Ser Leu Leu Cys LysAla Val 90 95 100 cac ttc ctc atc ttc ctc acc atg cac gcc agc agc ttcacg ctg gcc 452 His Phe Leu Ile Phe Leu Thr Met His Ala Ser Ser Phe ThrLeu Ala 105 110 115 gcc gtc tcc ctg gac agg tat ctg gcc atc cgc tac ccgctg cac tcc 500 Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg Tyr Pro LeuHis Ser 120 125 130 cgc gag ctg cgc acg cct cga aac gcg ctg gca gcc atcggg ctc atc 548 Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala Ala Ile GlyLeu Ile 135 140 145 tgg ggg ctg tcg ctg ctc ttc tcc ggg ccc tac ctg agctac tac cgc 596 Trp Gly Leu Ser Leu Leu Phe Ser Gly Pro Tyr Leu Ser TyrTyr Arg 150 155 160 165 cag tcg cag ctg gcc aac ctg acc gtg tgc cat cccgcg tgg agc gcc 644 Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His Pro AlaTrp Ser Ala 170 175 180 cct cgc cgc cgc gcc atg gac atc tgc acc ttc gtcttc agc tac ctg 692 Pro Arg Arg Arg Ala Met Asp Ile Cys Thr Phe Val PheSer Tyr Leu 185 190 195 ctt cct gtg ctg gtt ctc ggc ctg acc tac gcg cgcacc ttg cgc tac 740 Leu Pro Val Leu Val Leu Gly Leu Thr Tyr Ala Arg ThrLeu Arg Tyr 200 205 210 ctc tgg cgc gcc gtc gac ccg gtg gcc gcg ggc tcgggt gcc cgg cgc 788 Leu Trp Arg Ala Val Asp Pro Val Ala Ala Gly Ser GlyAla Arg Arg 215 220 225 gcc aag cgc aag gtg aca cgc atg atc ctc atc gtggcc gcg ctc ttc 836 Ala Lys Arg Lys Val Thr Arg Met Ile Leu Ile Val AlaAla Leu Phe 230 235 240 245 tgc ctc tgc tgg atg ccc cac cac gcg ctc atcctc tgc gtg tgg ttc 884 Cys Leu Cys Trp Met Pro His His Ala Leu Ile LeuCys Val Trp Phe 250 255 260 ggc cag ttc ccg ctc acg cgc gcc act tat gcgctt cgc atc ctc tcg 932 Gly Gln Phe Pro Leu Thr Arg Ala Thr Tyr Ala LeuArg Ile Leu Ser 265 270 275 cac ctg gtc tcc tac gcc aac tcc tgc gtc aacccc atc gtt tac gcg 980 His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn ProIle Val Tyr Ala 280 285 290 ctg gtc tcc aag cac ttc cgc aaa ggc ttc cgcacg atc tgc gcg ggc 1028 Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg ThrIle Cys Ala Gly 295 300 305 ctg ctg ggc cgt gcc cca ggc cga gcc tcg ggccgt gtg tgc gct gcc 1076 Leu Leu Gly Arg Ala Pro Gly Arg Ala Ser Gly ArgVal Cys Ala Ala 310 315 320 325 gcg cgg ggc acc cac agt ggc agc gtg ttggag cgc gag tcc agc gac 1124 Ala Arg Gly Thr His Ser Gly Ser Val Leu GluArg Glu Ser Ser Asp 330 335 340 ctg ttg cac atg agc gag gcg gcg ggg gccctt cgt ccc tgc ccc ggc 1172 Leu Leu His Met Ser Glu Ala Ala Gly Ala LeuArg Pro Cys Pro Gly 345 350 355 gct tcc cag cca tgc atc ctc gag ccc tgtcct ggc ccg tcc tgg cag 1220 Ala Ser Gln Pro Cys Ile Leu Glu Pro Cys ProGly Pro Ser Trp Gln 360 365 370 ggc cca aag gca ggc gac agc atc ctg acggtt gat gtg gcc tga 1265 Gly Pro Lys Ala Gly Asp Ser Ile Leu Thr Val AspVal Ala 375 380 385 aagcacttag cgggcgcgct gggatgtcac agagttggagtcattgttgg gggaccgtgg 1325 ggagagcttt gcctgttaat aaaacgcaca aaccatttca1365 5 387 PRT Homo sapiens 5 Met Asn Val Ser Gly Cys Pro Gly Ala GlyAsn Ala Ser Gln Ala Gly 1 5 10 15 Gly Gly Gly Gly Trp His Pro Glu AlaVal Ile Val Pro Leu Leu Phe 20 25 30 Ala Leu Ile Phe Leu Val Gly Thr ValGly Asn Thr Leu Val Leu Ala 35 40 45 Val Leu Leu Arg Gly Gly Gln Ala ValSer Thr Thr Asn Leu Phe Ile 50 55 60 Leu Asn Leu Gly Val Ala Asp Leu CysPhe Ile Leu Cys Cys Val Pro 65 70 75 80 Phe Gln Ala Thr Ile Tyr Thr LeuAsp Gly Trp Val Phe Gly Ser Leu 85 90 95 Leu Cys Lys Ala Val His Phe LeuIle Phe Leu Thr Met His Ala Ser 100 105 110 Ser Phe Thr Leu Ala Ala ValSer Leu Asp Arg Tyr Leu Ala Ile Arg 115 120 125 Tyr Pro Leu His Ser ArgGlu Leu Arg Thr Pro Arg Asn Ala Leu Ala 130 135 140 Ala Ile Gly Leu IleTrp Gly Leu Ser Leu Leu Phe Ser Gly Pro Tyr 145 150 155 160 Leu Ser TyrTyr Arg Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His 165 170 175 Pro AlaTrp Ser Ala Pro Arg Arg Arg Ala Met Asp Ile Cys Thr Phe 180 185 190 ValPhe Ser Tyr Leu Leu Pro Val Leu Val Leu Gly Leu Thr Tyr Ala 195 200 205Arg Thr Leu Arg Tyr Leu Trp Arg Ala Val Asp Pro Val Ala Ala Gly 210 215220 Ser Gly Ala Arg Arg Ala Lys Arg Lys Val Thr Arg Met Ile Leu Ile 225230 235 240 Val Ala Ala Leu Phe Cys Leu Cys Trp Met Pro His His Ala LeuIle 245 250 255 Leu Cys Val Trp Phe Gly Gln Phe Pro Leu Thr Arg Ala ThrTyr Ala 260 265 270 Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn SerCys Val Asn 275 280 285 Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe ArgLys Gly Phe Arg 290 295 300 Thr Ile Cys Ala Gly Leu Leu Gly Arg Ala ProGly Arg Ala Ser Gly 305 310 315 320 Arg Val Cys Ala Ala Ala Arg Gly ThrHis Ser Gly Ser Val Leu Glu 325 330 335 Arg Glu Ser Ser Asp Leu Leu HisMet Ser Glu Ala Ala Gly Ala Leu 340 345 350 Arg Pro Cys Pro Gly Ala SerGln Pro Cys Ile Leu Glu Pro Cys Pro 355 360 365 Gly Pro Ser Trp Gln GlyPro Lys Ala Gly Asp Ser Ile Leu Thr Val 370 375 380 Asp Val Ala 385 61384 DNA Homo sapiens 6 gtgagccagc gccttggcct ccctgggaga tgggcatccacgcgggggat ggagcgggag 60 gcgggactgg ggaccaagaa gggacgcgca gagtgggacaggacactaag aaggcagtgg 120 aagacaagcg ggcgcggagg aggaaaaaga ggaataagaatgggggaccg tggtgtccct 180 cggttagatg cgtcctgggg cctggaagcc tggagaatgtggctctccag cgccgcccgt 240 gcctgacaac gcgcagcgtt tcccagtacg acgcgtttgtgcgcgttcat ctcgcttgag 300 cttaatgccc tccgtgaggg tgggatagga caaagtgcccaatatacaga agagttgagt 360 tcctaagtaa ctcgctcaga gtcgccagcc agggatcgggtgcgtgaagt gaccgtctgt 420 ctcctgcagc caacttcagg cgcctccact gcgctcgcctccaagccacg gtttggttgg 480 ttggtgcagc tggctcaggt ccaggctgtg gatcttgggtcctttgcaag gatccactcc 540 ggagtcccag cgagcgtgcc taaaggtccc tagctcagtcccagcccact ctgcctctcg 600 cctccaaaca aaacaaaaca aaataaaatc caaaacaagtcggggccggg agaggagcgt 660 gccctggggt tcttcctccc cagccagagg agagcgagagacgcacattc gggagagcgc 720 gggactcagg tggagcttga aaggacactg ggatggttcctggggaggaa atccgggtat 780 ttcccctctc catcctctgg aaaaacagag aggcgaggccagactgcccc cacacctcct 840 gtagccactg agcgcgaagt gcgttggttc cgagcgcgctggtgggatcc acaaagctcg 900 cattctctca ggaatcccct gagaaattaa ctgtcccttgcccaacatgt cttctccagg 960 ctgtctgcta gagcctcagg cgcctccgcc ctccctcccgcggcaccgtc accagtgggt 1020 agtcacagcc tcccggagcc catagccggt tctccaacctttagtcttca gtggctttgg 1080 ggtgccctct cagtggagac tgtggttgca gtccccgggggcagcgggag aatggcttga 1140 aggcacacct ttcctgctgc cgggcccgcc ccatttccaggctccgctga gtgtctggga 1200 cacgctggga ggcccccacc tccgccctca cgccgagcctcacccccacc tcctctgtgt 1260 gcggtgtaac catgcgctaa ggaccttcct cgagagcagccttgggaccg aggtgcaggg 1320 gtcgcggccc tccagcatga atgtgcccgc tcagccgacgtctcccttcc cggtctgacc 1380 gcag 1384 7 346 PRT Rattus norvegicus 7 MetGlu Leu Ala Pro Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15Glu Pro Pro Ala Glu Pro Arg Pro Leu Phe Gly Ile Gly Val Glu Asn 20 25 30Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu 35 40 45Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Lys 50 55 60Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala Asp 65 70 7580 Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr Ala 85 9095 Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His Tyr 100105 110 Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala Met115 120 125 Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser SerSer 130 135 140 Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe IleTrp Ala 145 150 155 160 Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr TyrGln Arg Leu Phe 165 170 175 His Arg Asp Ser Asn Gln Thr Phe Cys Trp GluHis Trp Pro Asn Gln 180 185 190 Leu His Lys Lys Ala Tyr Val Val Cys ThrPhe Val Phe Gly Tyr Leu 195 200 205 Leu Pro Leu Leu Leu Ile Cys Phe CysTyr Ala Lys Val Leu Asn His 210 215 220 Leu His Lys Lys Leu Lys Asn MetSer Lys Lys Ser Glu Ala Ser Lys 225 230 235 240 Lys Lys Thr Ala Gln ThrVal Leu Val Val Val Val Val Phe Gly Ile 245 250 255 Ser Trp Leu Pro HisHis Val Ile His Leu Trp Ala Glu Phe Gly Ala 260 265 270 Phe Pro Leu ThrPro Ala Ser Phe Phe Phe Arg Ile Thr Ala His Cys 275 280 285 Leu Ala TyrSer Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe Leu 290 295 300 Ser GluAsn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys Arg Val 305 310 315 320Cys Asn Glu Ser Pro His Gly Asp Ala Lys Glu Lys Asn Arg Ile Asp 325 330335 Thr Pro Pro Ser Thr Asn Cys Thr His Val 340 345 8 24 DNA ArtificialSequence Description of Artificial Sequence primer 8 tgggcaacagcctagtgatc accg 24 9 24 DNA Artificial Sequence Description ofArtificial Sequence primer 9 ctgctcccag cagaaggtct ggtt 24 10 24 DNAArtificial Sequence Description of Artificial Sequence primer 10cctcagtgaa gggaatggga gcga 24 11 26 DNA Artificial Sequence Descriptionof Artificial Sequence primer 11 ctcattgcaa acacggcact tgaaca 26 12 24DNA Artificial Sequence Description of Artificial Sequence primer 12cttgcttgta cgccttccgg aagt 24 13 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 13 gagaacttca tcacgctggt ggtg 24 14 23 DNAArtificial Sequence Description of Artificial Sequence primer /probe 14ccctacctga gctactaccg tca 23 15 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer /probe 15 accaaaccac acgcagagga taag 24 1626 DNA Artificial Sequence Description of Artificial Sequence primer/probe 16 ccacgatgag gatcatgcgt gtcacc 26 17 26 DNA Artificial SequenceDescription of Artificial Sequence primer /probe 17 taggtcaggccgagaaccag cacagg 26 18 24 DNA Artificial Sequence Description ofArtificial Sequence primer /probe 18 caggtagctg aagacgaagg tgca 24 19 25DNA Artificial Sequence Description of Artificial Sequence primer /probe19 ctgcaccttc gtcttcagct acctg 25 20 26 DNA Artificial SequenceDescription of Artificial Sequence primer /probe 20 cctgtgctggttctcggcct gaccta 26 21 26 DNA Artificial Sequence Description ofArtificial Sequence primer /probe 21 tatctggcca tccgctaccc gctgca 26 2245 DNA Artificial Sequence Description of Artificial Sequence primer/probe 22 ttgcgctacc tctggcgcgc cgtcgacccg gtggccgcgg gctcg 45 23 25 DNAArtificial Sequence Description of Artificial Sequence primer /probe 23ccaacaatga ctccaactct gtgac 25 24 25 DNA Artificial Sequence Descriptionof Artificial Sequence primer /probe 24 aggcgcagaa ctggtaggta tggaa 2525 25 DNA Artificial Sequence Description of Artificial Sequence primer/probe 25 aagcttctag agatccctcg acctc 25 26 28 DNA Artificial SequenceDescription of Artificial Sequence primer 26 acggaattcg acatgaatggctccggca 28 27 34 DNA Artificial Sequence Description of ArtificialSequence primer 27 gctctagagc ccctttggtc ctttaacaag ccgg 34 28 26 DNAArtificial Sequence Description of Artificial Sequence primer 28caaggctgtt catttcctca tctttc 26 29 25 DNA Artificial SequenceDescription of Artificial Sequence primer 29 ttggagacca gagcgtaaac gatgg25 30 45 DNA Artificial Sequence Description of Artificial Sequenceprimer 30 agtcgacccg gtgactgcag gctcaggttc ccagcgcgcc aaacg 45

What is claimed is:
 1. A nucleic acid encoding a GALR2 receptor.
 2. Thenucleic acid of claim 1, wherein the nucleic acid is DNA.
 3. The DNA ofclaim 2, wherein the DNA is cDNA.
 4. The DNA of claim 2, wherein the DNAis genomic DNA.
 5. The nucleic acid of claim 1, wherein the nucleic acidis RNA.
 6. The nucleic acid of claim 1, wherein the nucleic acid encodesa vertebrate GALR2 receptor.
 7. The nucleic acid of claim 1, wherein thenucleic acid encodes a mammalian GALR2 receptor.
 8. The nucleic acid ofclaim 1, wherein the nucleic acid encodes a rat GALR2 receptor.
 9. Thenucleic acid of claim 1, wherein the nucleic acid encodes a human GALR2receptor.
 10. The nucleic acid of claim 7, wherein the nucleic acidencodes a receptor characterized by an amino acid sequence in thetransmembrane region which has a homology of 60% or higher to the aminoacid sequence in the transmembrane region of the rat GALR2 receptor anda homology of less than 60% to the amino acid sequence in thetransmembrane region of any GALR1 receptor.
 11. The nucleic acid ofclaim 7, wherein the nucleic acid encodes a mammalian GALR2 receptorwhich has substantially the same amino acid sequence as does the GALR2receptor encoded by the plasmid K985 (ATCC Accession No. 97426).
 12. Thenucleic acid of claim 8, wherein the nucleic acid encodes a rat GALR2receptor which has an amino acid sequence encoded by the plasmid K985(ATCC Accession No. 97426).
 13. The nucleic acid of claim 7, wherein thenucleic acid encodes a mammalian GALR2 receptor which has substantiallythe same amino acid sequence as does the GALR2 receptor encoded by theplasmid K1045 (ATCC Accession No. 97778).
 14. The nucleic acid of claim8, wherein the nucleic acid encodes a rat GALR2 receptor which has anamino acid sequence encoded by the plasmid K1045 (ATCC Accession No.97778).
 15. The nucleic acid of claim 8, wherein the nucleic acidencodes a rat GALR2 receptor which has substantially the same amino acidsequence as that shown in FIG.
 2. 16. The nucleic acid of claim 8,wherein the nucleic acid encodes a rat GALR2 receptor which has theamino acid sequence shown in FIG.
 2. 17. The nucleic acid of claim 7,wherein the nucleic acid encodes a mammalian GALR2 receptor which hassubstantially the same amino acid sequence as does the GALR2 receptorencoded by the plasmid BO29 (ATCC Accession No. 97735).
 18. The nucleicacid of claim 9, wherein the nucleic acid encodes a human GALR2 receptorwhich has an amino acid sequence encoded by the plasmid BO29 (ATCCAccession No. 97735).
 19. The nucleic acid of claim 7, wherein thenucleic acid encodes a mammalian GALR2 receptor which has substantiallythe same amino acid sequence as does the GALR2 receptor encoded by theplasmid BO39 (ATCC Accession No. ______).
 20. The nucleic acid of claim9, wherein the nucleic acid encodes a human GALR2 receptor which has anamino acid sequence encoded by the plasmid BO39 (ATCC Accession No.______).
 21. The nucleic acid of claim 9, wherein the nucleic acidencodes a human GALR2 receptor which has substantially the same aminoacid sequence as that shown in FIG.
 11. 22. The nucleic acid of claim 9,wherein the nucleic acid encodes a human GALR2 receptor which has theamino acid sequence shown in FIG.
 11. 23. An isolated nucleic acidencoding a modified GALR2 receptor, which differs from a GALR2 receptorby having an amino acid(s) deletion, replacement or addition in thethird intracellular domain.
 24. The nucleic acid of claim 23, whereinthe modified GALR2 receptor differs from a GALR2 receptor by having adeletion in the third intracellular domain.
 25. The nucleic acid ofclaim 23, wherein the modified GALR2 receptor differs from a GALR2receptor by having a replacement or addition in the third intracellulardomain.
 26. A purified GALR2 receptor protein.
 27. A vector comprisingthe nucleic acid of claim
 1. 28. A vector comprising the nucleic acid ofeither of claims 8 or
 9. 29. A vector of claim 27 adapted for expressionin a bacterial cell which comprises the regulatory elements necessaryfor expression of the nucleic acid in the bacterial cell operativelylinked to the nucleic acid encoding a GALR2 receptor as to permitexpression thereof.
 30. A vector of claim 27 adapted for expression in ayeast cell which comprises the regulatory elements necessary forexpression of the nucleic acid in the yeast cell operatively linked tothe nucleic acid encoding a GALR2 receptor as to permit expressionthereof.
 31. A vector of claim 27 adapted for expression in an insectcell which comprises the regulatory elements necessary for expression ofthe nucleic acid in the insect cell operatively linked to the nucleicacid encoding the GALR2 receptor as to permit expression thereof.
 32. Avector of claim 31 which is a baculovirus.
 33. A vector of claim 27adapted for expression in a mammalian cell which comprises theregulatory elements necessary for expression of the nucleic acid in themammalian cell operatively linked to the nucleic acid encoding a GALR2receptor as to permit expression thereof.
 34. A vector of claim 27wherein the vector is a plasmid.
 35. The plasmid of claim 34 designatedK985 (ATCC Accession No. 97426).
 36. The plasmid of claim 34 designatedK1045 (ATCC Accession No. 97778).
 37. The plasmid of claim 34 designatedBO29 (ATCC Accession No. 97735).
 38. The plasmid of claim 34 designatedBO39 (ATCC Accession No. ______).
 39. A cell comprising the vector ofclaim
 27. 40. A cell of claim 39, wherein the cell is a non-mammaliancell.
 41. A cell of claim 40, wherein the non-mammalian cell is aXenopus oocyte cell or a Xenopus melanophore cell.
 42. A cell of claim39, wherein the cell is a mammalian cell.
 43. A mammalian cell of claim42, wherein the cell is a COS-7 cell, a 293 human embryonic kidney cell,a NIH-3T3 cell, a LM(tk−) cell or a CHO cell.
 44. The LM(tk−) cell ofclaim 43 designated L-rGALR2-8 (ATCC Accession No. CRL-12074).
 45. TheLM(tk−) cell of claim 43 designated L-rGALR2I-4 (ATCC Accession No.CRL-12223).
 46. The CHO cell of claim 43 designated C-rGalR2-79 (ATCCAccession No. ______).
 47. An insect cell comprising the vector of claim31.
 48. An insect cell of claim 47, wherein the insect cell is an Sf9cell.
 49. An insect cell of claim 47, wherein the insect cell is an Sf21cell.
 50. A membrane preparation isolated from the cell of either ofclaims 39 or
 47. 51. A nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid K985. 52.A nucleic acid probe comprising at least 15 nucleotides, which probespecifically hybridizes with a nucleic acid encoding a GALR2 receptor,wherein the probe has a unique sequence corresponding to a sequencepresent within one of the two strands of the nucleic acid encoding theGALR2 receptor contained in plasmid K1045.
 53. A nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a GALR2 receptor, wherein the probe has aunique sequence corresponding to a sequence present within (a) thenucleic acid sequence shown in FIG. 1 or (b) the reverse complement ofthe nucleic acid sequence shown in FIG.
 1. 54. A nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a GALR2 receptor, wherein the probe has aunique sequence corresponding to a sequence present within one of thetwo strands of the nucleic acid encoding the GALR2 receptor contained inplasmid BO29.
 55. A nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a GALR2 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding the GALR2 receptor contained in plasmid BO39. 56.A nucleic acid probe comprising at least 15 nucleotides, which probespecifically hybridizes with a nucleic acid encoding a GALR2 receptor,wherein the probe has a unique sequence corresponding to a sequencepresent within (a) the nucleic acid sequence shown in FIG. 10 or (b) thereverse complement of the nucleic acid sequence shown in FIG.
 10. 57.The nucleic acid probe of any one of claims 51, 52, 53, 54, 55 or 56,wherein the nucleic acid is DNA.
 58. The nucleic acid probe of any oneof claims 51, 52, 53, 54, 55 or 56 wherein the nucleic acid is RNA. 59.A nucleic acid probe comprising a nucleic acid molecule of at least 15nucleotides which is complementary to a unique fragment of the sequenceof a nucleic acid molecule encoding a GALR2 receptor.
 60. A nucleic acidprobe comprising a nucleic acid molecule of at least 15 nucleotideswhich is complementary to the antisense sequence of a unique fragment ofthe sequence of a nucleic acid molecule encoding a GALR2 receptor. 61.An antisense oligonucleotide having a sequence capable of specificallyhybridizing to the mRNA of claim 5, so as to prevent translation of themRNA.
 62. An antisense oligonucleotide having a sequence capable ofspecifically hybridizing to the genomic DNA of claim
 4. 63. An antisenseoligonucleotide of either of claims 61 or 62, wherein theoligonucleotide comprises chemically modified nucleotides or nucleotideanalogues.
 64. An antibody capable of binding to a GALR2 receptorencoded by the nucleic acid of claim
 1. 65. The antibody of claim 64,wherein the GALR2 receptor is a human GALR2 receptor.
 66. An antibodycapable of competitively inhibiting the binding of the antibody of claim64 to a GALR2 receptor.
 67. An antibody of claim 64, wherein theantibody is a monoclonal antibody.
 68. A monoclonal antibody of claim 67directed to an epitope of a GALR2 receptor present on the surface of aGALR2 receptor expressing cell.
 69. A pharmaceutical compositioncomprising an amount of the oligonucleotide of claim 61 capable ofpassing through a cell membrane effective to reduce expression of aGALR2 receptor and a pharmaceutically acceptable carrier capable ofpassing through a cell membrane.
 70. A pharmaceutical composition ofclaim 69, wherein the oligonucleotide is coupled to a substance whichinactivates mRNA.
 71. A pharmaceutical composition of claim 70, whereinthe substance which inactivates mRNA is a ribozyme.
 72. A pharmaceuticalcomposition of claim 69, wherein the pharmaceutically acceptable carriercomprises a structure which binds to a receptor on a cell capable ofbeing taken up by the cells after binding to the structure.
 73. Apharmaceutical composition of claim 72, wherein the pharmaceuticallyacceptable carrier is capable of binding to a receptor which is specificfor a selected cell type.
 74. A pharmaceutical composition whichcomprises an amount of the antibody of claim 64 effective to blockbinding of a ligand to the GALR2 receptor and a pharmaceuticallyacceptable carrier.
 75. A transgenic nonhuman mammal expressing DNAencoding a GALR2 receptor of claim
 1. 76. A transgenic nonhuman mammalcomprising a homologous recombination knockout of the native GALR2receptor.
 77. A transgenic nonhuman mammal whose genome comprisesantisense DNA complementary to DNA encoding a GALR2 receptor of claim 1so placed as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding a GALR2 receptor and which hybridizes tomRNA encoding a GALR2 receptor, thereby reducing its translation. 78.The transgenic nonhuman mammal of claim 75 or 76, wherein the DNAencoding a GALR2 receptor additionally comprises an inducible promoter.79. The transgenic nonhuman mammal of claim 75 or 77, wherein the DNAencoding a GALR2 receptor additionally comprises tissue specificregulatory elements.
 80. A transgenic nonhuman mammal of any one ofclaims 75, 76 or 77, wherein the transgenic nonhuman mammal is a mouse.81. A process for identifying a chemical compound which specificallybinds to a GALR2 receptor which comprises contacting cells containingDNA encoding and expressing on their cell surface the GALR2 receptor,wherein such cells do not normally express the GALR2 receptor, with thecompound under conditions suitable for binding, and detecting specificbinding of the chemical compound to the GALR2 receptor.
 82. A processfor identifying a chemical compound which specifically binds to a GALR2receptor which comprises contacting a membrane fraction from a cellextract of cells containing DNA encoding and expressing on their cellsurface the GALR2 receptor, wherein such cells do not normally expressthe GALR2 receptor, with the compound under conditions suitable forbinding, and detecting specific binding of the chemical compound to theGALR2 receptor.
 83. The process of claim 81 or 82, wherein the GALR2receptor is a mammalian GALR2 receptor.
 84. The process of claim 81 or82, wherein the GALR2 receptor has substantially the same amino acidsequence as encoded by the plasmid K985 (ATCC Accession No. 97426). 85.The process of claim 81 or 82, wherein the GALR2 receptor hassubstantially the same amino acid sequence as that shown in FIG. 2 (Seq.ID No. 8).
 86. The process of claim 81 or 82, wherein the GALR2 receptorhas substantially the same amino acid sequence as encoded by the plasmidBO29 (ATCC Accession No. 97735).
 87. The process of claim 81 or 82,wherein the GALR2 receptor has substantially the same amino acidsequence as that shown in FIG. 11 (Seq. ID No. 30).
 88. The method ofany one of claims 81, 82, 83, 84, 85, 86 or 87, wherein the compound isnot previously known to bind to a GALR2 receptor.
 89. A compounddetermined by the method of claim
 88. 90. A process for determiningwhether a chemical compound is a GALR2 receptor agonist which comprisescontacting cells transfected with and expressing DNA encoding the GALR2receptor with the compound under conditions permitting the activation ofthe GALR2 receptor, and detecting an increase in GALR2 receptoractivity, so as to thereby determine whether the compound is a GALR2receptor agonist.
 91. A process for determining whether a chemicalcompound is a GALR2 receptor agonist which comprises preparing a cellextract from cells transfected with and expressing DNA encoding theGALR2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with the compound under conditionspermitting the activation of the GALR2 receptor, and detecting anincrease in GALR2 receptor activity, so as to thereby determine whetherthe compound is a GALR2 receptor agonist.
 92. The process of claim 90 or91, wherein the GALR2 receptor is a mammalian GALR2 receptor.
 93. Theprocess of claim 90 or 91, wherein the GALR2 receptor has substantiallythe same amino acid sequence as encoded by the plasmid K985 (ATCCAccession No. 97426).
 94. The process of claim 90 or 91, wherein theGALR2 receptor has substantially the same amino acid sequence as thatshown in FIG. 2 (Seq. ID No. 8).
 95. The process of claim 90 or 91,wherein the GALR2 receptor has substantially the same amino acidsequence as encoded by the plasmid BO29 (ATCC Accession No. 97735). 96.The process of claim 90 or 91, wherein the GALR2 receptor hassubstantially the same amino acid sequence as that shown in FIG. 11(Seq. ID No. 30).
 97. A process for determining whether a chemicalcompound is a GALR2 receptor antagonist which comprises contacting cellstransfected with and expressing DNA encoding the GALR2 receptor with thecompound in the presence of a known GALR2 receptor agonist, underconditions permitting the activation of the GALR2 receptor, anddetecting a decrease in GALR2 receptor activity, so as to therebydetermine whether the compound is a GALR2 receptor antagonist.
 98. Aprocess for determining whether a chemical compound is a GALR2 receptorantagonist which comprises preparing a cell extract from cellstransfected with and expressing DNA encoding the GALR2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with the ligand in the presence of a known GALR2receptor agonist, under conditions permitting the activation of theGALR2 receptor, and detecting a decrease in GALR2 receptor activity, soas to thereby determine whether the compound is a GALR2 receptorantagonist.
 99. The process of claim 97 or 98, wherein the GALR2receptor is a mammalian GALR2 receptor.
 100. The process of claim 97 or98, wherein the GALR2 receptor has substantially the same amino acidsequence as encoded by the plasmid K985 (ATCC Accession No. 97426). 101.The process of claim 97 or 98, wherein the GALR2 receptor hassubstantially the same amino acid sequence as that shown in FIG. 2 (Seq.ID No. 8).
 102. The process of claim 97 or 98, wherein the GALR2receptor has substantially the same amino acid sequence as encoded bythe plasmid BO29 (ATCC Accession No. 97735).
 103. The process of claim97 or 98, wherein the GALR2 receptor has substantially the same aminoacid sequence as that shown in FIG. 11 (Seq. ID No. 30).
 104. Theprocess of any one of claims 81, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102 or 103, wherein the cell is aninsect cell.
 105. The process of any one of claims 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 or103, wherein the cell is a mammalian cell.
 106. The process of claim105, wherein the cell is nonneuronal in origin.
 107. The process ofclaim 106, wherein the nonneuronal cell is a COS-7 cell, 293 humanembryonic kidney cell, NIH-3T3 cell or LM(tk−) cell.
 108. The process ofclaim 106, wherein the nonneuronal cell is the LM(tk−) cell designatedL-rGALR2-8 (ATCC Accession No. CRL-12074).
 109. The process of claim106, wherein the nonneuronal cell is the LM(tk−) cell designatedL-rGALR2I-4 (ATCC Accession No. CRL-12223).
 110. The process of claim106, wherein the nonneuronal cell is the CHO cell designated C-rGalR2-79(ATCC Accession No. ______).
 111. The process of claim 105 wherein thecompound is not previously known to bind to a GALR2 receptor.
 112. Acompound determined by the process of claim
 111. 113. A pharmaceuticalcomposition which comprises an amount of a GALR2 receptor agonistdetermined by the process of claim 90 or 91 effective to increaseactivity of a GALR2 receptor and a pharmaceutically acceptable carrier.114. A pharmaceutical composition of claim 113, wherein the GALR2receptor agonist is not previously known.
 115. A pharmaceuticalcomposition which comprises an amount of a GALR2 receptor antagonistdetermined by the process of claim 97 or 98 effective to reduce activityof a GALR2 receptor and a pharmaceutically acceptable carrier.
 116. Apharmaceutical composition of claim 115, wherein the GALR2 receptorantagonist is not previously known.
 117. A process involving competitivebinding for identifying a chemical compound which specifically binds toa GALR2 receptor which comprises separately contacting cells expressingon their cell surface the GALR2 receptor, wherein such cells do notnormally express the GALR2 receptor, with both the chemical compound anda second chemical compound known to bind to the receptor, and with onlythe second chemical compound, under conditions suitable for binding ofboth compounds, and detecting specific binding of the chemical compoundto the GALR2 receptor, a decrease in the binding of the second chemicalcompound to the GALR2 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the GALR2 receptor. 118.A process involving competitive binding for identifying a chemicalcompound which specifically binds to a human GALR2 receptor whichcomprises separately contacting a membrane fraction from a cell extractof cells expressing on their cell surface the GALR2 receptor, whereinsuch cells do not normally express the GALR2 receptor, with both thechemical compound and a second chemical compound known to bind to thereceptor, and with only the second chemical compound, under conditionssuitable for binding of both compounds, and detecting specific bindingof the chemical compound to the GALR2 receptor, a decrease in thebinding of the second chemical compound to the GALR2 receptor in thepresence of the chemical compound indicating that the chemical compoundbinds to the GALR2 receptor.
 119. A process for determining whether achemical compound specifically binds to and activates a GALR2 receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the GALR2 receptor, wherein suchcells do not normally express the GALR2 receptor, with the chemicalcompound under conditions suitable for activation of the GALR2 receptor,and measuring the second messenger response in the presence and in theabsence of the chemical compound, a change in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the GALR2 receptor.
 120. A process for determiningwhether a chemical compound specifically binds to and activates a GALR2receptor, which comprises contacting a membrane fraction isolated from acell extract of cells producing a second messenger response andexpressing on their cell surface the GALR2 receptor, wherein such cellsdo not normally express the GALR2 receptor, with the chemical compoundunder conditions suitable for activation of the GALR2 receptor, andmeasuring the second messenger response in the presence and in theabsence of the chemical compound, a change in the second messengerresponse in the presence of the chemical compound indicating that thecompound activates the GALR2 receptor.
 121. A process for determiningwhether a chemical compound specifically binds to and inhibitsactivation of a GALR2 receptor, which comprises separately contactingcells producing a second messenger response and expressing on their cellsurface the GALR2 receptor, wherein such cells do not normally expressthe GALR2 receptor, with both the chemical compound and a secondchemical compound known to activate the GALR2 receptor, and with onlythe second chemical compound, under conditions suitable for activationof the GALR2 receptor, and measuring the second messenger response inthe presence of only the second chemical compound and in the presence ofboth the second chemical compound and the chemical compound, a smallerchange in the second messenger response in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound indicating that the chemicalcompound inhibits activation of the GALR2 receptor.
 122. A process fordetermining whether a chemical compound specifically binds to andinhibits activation of a GALR2 receptor, which comprises separatelycontacting a membrane fraction from a cell extract of cells producing asecond messenger response and expressing on their cell surface the GALR2receptor, wherein such cells do not normally express the GALR2 receptor,with both the chemical compound and a second chemical compound known toactivate the GALR2 receptor, and with only the second chemical compound,under conditions suitable for activation of the GALR2 receptor, andmeasuring the second messenger response in the presence of only thesecond chemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the GALR2 receptor.
 123. The process of claim 119 or 120,wherein the second messenger response comprises arachidonic acid releaseand the change in second messenger response is an increase inarachidonic acid levels.
 124. The process of claim 119 or 120, whereinthe second messenger response comprises intracellular calcium levels andthe change in second messenger response is an increase in intracellularcalcium levels.
 125. The process of claim 119 or 120, wherein the secondmessenger response comprises inositol phospholipid hydrolysis and thechange in second messenger response is an increase in inositolphospholipid hydrolysis.
 126. The process of claim 121 or 122, whereinthe second messenger response comprises arachidonic acid release and thechange in second messenger response is a smaller increase in the levelof arachidonic acid in the presence of both the chemical compound andthe second chemical compound than in the presence of only the secondchemical compound.
 127. The process of claim 121 or 122, wherein thesecond messenger response comprises intracellular calcium levels, andthe change in second messenger response is a smaller increase in theintracellular calcium levels in the presence of both the chemicalcompound and the second chemical compound than in the presence of onlythe second chemical compound.
 128. The process of claim 121 or 122,wherein the second messenger response comprises inositol phospholipidhydrolysis, and the change in second messenger response is a smallerincrease in inositol phospholipid hydrolysis in the presence of both thechemical compound and the second chemical compound than in the presenceof only the second chemical compound.
 129. A process of any one ofclaims 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 or 128,wherein the GALR2 receptor is a mammalian GALR2 receptor.
 130. Theprocess of claim 129, wherein the GALR2 receptor has substantially thesame amino acid sequence as encoded by the plasmid K985 ATCC AccessionNo. 97426).
 131. The process of claim 129, wherein the GALR2 receptorhas substantially the same amino acid sequence as that shown in FIG. 2(Seq. ID No. 8).
 132. The process of claim 129, wherein the GALR2receptor has substantially the same amino acid sequence as encoded bythe plasmid BO29 (ATCC Accession No. 97735).
 133. The process of claim129, wherein the GALR2 receptor has substantially the same amino acidsequence as that shown in FIG. 11 (Seq. ID No. 30).
 134. The process ofany one of claims 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132 or 133, wherein the cell is an insect cell. 135.The process of any one of claims 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132 or 133, wherein the cell is amammalian cell.
 136. The process of claim 135, wherein the mammaliancell is nonneuronal in origin.
 137. The process of claim 136, whereinthe nonneuronal cell is a COS-7 cell, 293 human embryonic kidney cell,NIH-3T3 cell or LM(tk−) cell.
 138. The process of claim 136, wherein thenonneuronal cell is the LM(tk−) cell designated L-rGALR2-8 (ATCCAccession No. CRL-12074).
 139. The process of claim 136, wherein thenonneuronal cell is the LM(tk−) cell designated L-rGALR2I-4 (ATCCAccession No. CRL-12223).
 140. The process of claim 136, wherein thenonneuronal cell is the CHO cell designated C-rGalR2-79 (ATCC AccessionNo. ______).
 141. The process of claim 135, wherein the compound is notpreviously known to bind to a GALR2 receptor.
 142. A compound determinedby the process of claim
 141. 143. A pharmaceutical composition whichcomprises an amount of a GALR2 receptor agonist determined by theprocess of any one of claims 119, 120, 123, 124, or 125 effective toincrease activity of a GALR2 receptor and a pharmaceutically acceptablecarrier.
 144. A pharmaceutical composition of claim 143, wherein theGALR2 receptor agonist is not previously known.
 145. A pharmaceuticalcomposition which comprises an amount of a GALR2 receptor antagonistdetermined by the process of any one of claims 121, 122, 126, 127 or 128effective to reduce activity of a GALR2 receptor and a pharmaceuticallyacceptable carrier.
 146. A pharmaceutical composition of claim 145,wherein the GALR2 receptor antagonist is not previously known.
 147. Amethod of screening a plurality of chemical compounds not known to bindto a GALR2 receptor to identify a compound which specifically binds tothe GALR2 receptor, which comprises (a) contacting cells transfectedwith and expressing DNA encoding the GALR2 receptor with a compoundknown to bind specifically to the GALR2 receptor; (b) contacting thepreparation of step (a) with the plurality of compounds not known tobind specifically to the GALR2 receptor, under conditions permittingbinding of compounds known to bind the GALR2 receptor; (c) determiningwhether the binding of the compound known to bind to the GALR2 receptoris reduced in the presence of the compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the GALR2 receptor of eachcompound included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the GALR2 receptor.148. A method of screening a plurality of chemical compounds not knownto bind to a GALR2 receptor to identify a compound which specificallybinds to the GALR2 receptor, which comprises (a) preparing a cellextract from cells transfected with and expressing DNA encoding theGALR2 receptor, isolating a membrane fraction from the cell extract,contacting the membrane fraction with a compound known to bindspecifically to the GALR2 receptor; (b) contacting the preparation ofstep (a) with the plurality of compounds not known to bind specificallyto the GALR2 receptor, under conditions permitting binding of compoundsknown to bind the GALR2 receptor; (c) determining whether the binding ofthe compound known to bind to the GALR2 receptor is reduced in thepresence of the compounds, relative to the binding of the compound inthe absence of the plurality of compounds; and if so (d) separatelydetermining the binding to the GALR2 receptor of each compound includedin the plurality of compounds, so as to thereby identify the compoundwhich specifically binds to the GALR2 receptor.
 149. A method of claim147 or 148, wherein the GALR2 receptor is a mammalian GALR2 receptor.150. A method of screening a plurality of chemical compounds not knownto activate a GALR2 receptor to identify a compound which activates theGALR2 receptor which comprises (a) contacting cells transfected with andexpressing the GALR2 receptor with the plurality of compounds not knownto activate the GALR2 receptor, under conditions permitting activationof the GALR2 receptor; (b) determining whether the activity of the GALR2receptor is increased in the presence of the compounds; and if so (c)separately determining whether the activation of the GALR2 receptor isincreased by each compound included in the plurality of compounds, so asto thereby identify the compound which activates the GALR2 receptor.151. A method of screening a plurality of chemical compounds not knownto activate a GALR2 receptor to identify a compound which activates theGALR2 receptor which comprises (a) preparing a cell extract from cellstransfected with and expressing DNA encoding the GALR2 receptor,isolating a membrane fraction from the cell extract, contacting themembrane fraction with the plurality of compounds not known to activatethe GALR2 receptor, under conditions permitting activation of the GALR2receptor; (b) determining whether the activity of the GALR2 receptor isincreased in the presence of the compounds; and if so (c) separatelydetermining whether the activation of the GALR2 receptor is increased byeach compound included in the plurality of compounds, so as to therebyidentify the compound which activates the GALR2 receptor.
 152. A methodof claim 150 or 151, wherein the GALR2 receptor is a mammalian GALR2receptor.
 153. A method of screening a plurality of chemical compoundsnot known to inhibit the activation of a GALR2 receptor to identify acompound which inhibits the activation of the GALR2 receptor, whichcomprises (a) contacting cells transfected with and expressing the GALR2receptor with the plurality of compounds in the presence of a knownGALR2 receptor agonist, under conditions permitting activation of theGALR2 receptor; (b) determining whether the activation of the GALR2receptor is reduced in the presence of the plurality of compounds,relative to the activation of the GALR2 receptor in the absence of theplurality of compounds; and if so (c) separately determining theinhibition of activation of the GALR2 receptor for each compoundincluded in the plurality of compounds, so as to thereby identify thecompound which inhibits the activation of the GALR2 receptor.
 154. Amethod of screening a plurality of chemical compounds not known toinhibit the activation of a GALR2 receptor to identify a compound whichinhibits the activation of the GALR2 receptor, which comprises (a)preparing a cell extract from cells transfected with and expressing DNAencoding the GALR2 receptor, isolating a membrane fraction from the cellextract, contacting the membrane fraction with the plurality ofcompounds in the presence of a known GALR2 receptor agonist, underconditions permitting activation of the GALR2 receptor; (b) determiningwhether the activation of the GALR2 receptor is reduced in the presenceof the plurality of compounds, relative to the activation of the GALR2receptor in the absence of the plurality of compounds; and if so (c)separately determining the inhibition of activation of the GALR2receptor for each compound included in the plurality of compounds, so asto thereby identify the compound which inhibits the activation of theGALR2 receptor.
 155. A method of any one of claims 90, 91, 97, 98, 150,151, 153, or 154, wherein activation of the GALR2 receptor is determinedby a second messenger assay.
 156. The method of claim 155, wherein thesecond messenger is cyclic AMP, intracellular calcium, or an inositolphospholipid.
 157. A method of claim 153 or 154, wherein the GALR2receptor is a mammalian GALR2 receptor.
 158. A method of any one ofclaims 147, 148, 150, 151, 153, or 154, wherein the cell is a mammaliancell.
 159. A method of claim 158, wherein the mammalian cell isnon-neuronal in origin.
 160. The method of claim 159, wherein thenon-neuronal cell is a COS-7 cell, a 293 human embryonic kidney cell, aLM(tk−) cell or an NIH-3T3 cell.
 161. A pharmaceutical compositioncomprising a compound identified by the method of claim 150 and apharmaceutically acceptable carrier.
 162. A pharmaceutical compositioncomprising a compound identified by the method of claim 153 and apharmaceutically acceptable carrier.
 163. A method of detectingexpression of a GALR2 receptor by detecting the presence of mRNA codingfor the GALR2 receptor which comprises obtaining total mRNA from thecell and contacting the mRNA so obtained with the nucleic acid probe ofany one of claims 51, 52, 53, 54, 55, 56 or 60 under hybridizingconditions, detecting the presence of mRNA hybridized to the probe, andthereby detecting the expression of the GALR2 receptor by the cell. 164.A method of treating an abnormality in a subject, wherein theabnormality is alleviated by the inhibition of a GALR2 receptor whichcomprises administering to a subject an effective amount of thepharmaceutical composition of any one of claims 115, 116, or 162effective to decrease the activity of the GALR2 receptor in the subject,thereby treating the abnormality in the subject.
 165. The method ofclaim 164, wherein the abnormality is obesity or bulimia.
 166. A methodof treating an abnormality in a subject wherein the abnormality isalleviated by the activation of a GALR2 receptor which comprisesadministering to a subject an effective amount of the pharmaceuticalcomposition of any one of claims 113, 114, or 161 effective to activatethe GALR2 receptor in the subject.
 167. The method of claim 166, whereinthe abnormal condition is anorexia.
 168. The method of claim 164 or 166,wherein the compound binds selectively to a GALR2 receptor.
 169. Themethod of claim 168, wherein the compound binds to the GALR2 receptorwith an affinity greater than ten-fold higher than the affinity withwhich the compound binds to a GALR1 receptor.
 170. The method of claim168, wherein the compound binds to the GALR2 receptor with an affinitygreater than ten-fold higher than the affinity with which the compoundbinds to a GALR3 receptor.
 171. A method of detecting the presence of aGALR2 receptor on the surface of a cell which comprises contacting thecell with the antibody of claim 64 under conditions permitting bindingof the antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of a GALR2receptor on the surface of the cell.
 172. A method of determining thephysiological effects of varying levels of activity of GALR2 receptorswhich comprises producing a transgenic nonhuman mammal of claim 78 whoselevels of GALR2 receptor activity are varied by use of an induciblepromoter which regulates GALR2 receptor expression.
 173. A method ofdetermining the physiological effects of varying levels of activity ofGALR2 receptors which comprises producing a panel of transgenic nonhumanmammals of claim 78 each expressing a different amount of GALR2receptor.
 174. A method for identifying an antagonist capable ofalleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a GALR2 receptor comprising administering acompound to the transgenic nonhuman mammal of any one of claims 75, 78,79, or 80, and determining whether the compound alleviates the physicaland behavioral abnormalities displayed by the transgenic nonhuman mammalas a result of overactivity of a GALR2 receptor, the alleviation of theabnormality identifying the compound as an antagonist.
 175. Anantagonist identified by the method of claim
 174. 176. A pharmaceuticalcomposition comprising an antagonist identified by the method of claim174 and a pharmaceutically acceptable carrier.
 177. A method of treatingan abnormality in a subject wherein the abnormality is alleviated bydecreasing the activity of a GALR2 receptor which comprisesadministering to a subject an effective amount of the pharmaceuticalcomposition of claim 176, thereby treating the abnormality.
 178. Amethod for identifying an agonist capable of alleviating an abnormalityin a subject wherein the abnormality is alleviated by increasing theactivity of a GALR2 receptor comprising administering a compound to thetransgenic nonhuman mammal of any one of claims 75, 78, 79, or 80, anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic nonhuman mammal, thealleviation of the abnormality identifying the compound as an agonist.179. An agonist identified by the method of claim
 178. 180. Apharmaceutical composition comprising an agonist identified by themethod of claim 178 and a pharmaceutically acceptable carrier.
 181. Amethod for treating an abnormality in a subject wherein the abnormalityis alleviated by increasing the activity of a GALR2 receptor whichcomprises administering to a subject an effective amount of thepharmaceutical composition of claim 180, thereby treating theabnormality.
 182. A method for diagnosing a predisposition to a disorderassociated with the activity of a specific human GALR2 receptor allelewhich comprises: a. obtaining DNA of subjects suffering from thedisorder; b. performing a restriction digest of the DNA with a panel ofrestriction enzymes; c. electrophoretically separating the resulting DNAfragments on a sizing gel; d. contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human GALR2 receptor and labelled with a detectable marker;e. detecting labelled bands which have hybridized to the DNA encoding ahuman GALR2 receptor of claim 9 labelled with a detectable marker tocreate a unique band pattern specific to the DNA of subjects sufferingfrom the disorder; f. preparing DNA obtained for diagnosis by steps a-e;and g. comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step e and the DNA obtained fordiagnosis from step f to determine whether the patterns are the same ordifferent and to diagnose thereby predisposition to the disorder if thepatterns are the same.
 183. The method of claim 182, wherein a disorderassociated with the activity of a specific human GALR2 receptor alleleis diagnosed.
 184. A method of preparing the purified GALR2 receptor ofclaim 26, which comprises: a. inducing cells to express GALR2 receptor;b. recovering the receptor from the induced cells; and c. purifying thereceptor so recovered.
 185. A method of preparing the purified GALR2receptor of claim 26, which comprises: a. inserting nucleic acidencoding the GALR2 receptor in a suitable vector; b. introducing theresulting vector in a suitable host cell; c. placing the resulting cellin suitable condition permitting the production of the isolated GALR2receptor; d. recovering the receptor produced by the resulting cell; ande. purifying the receptor so recovered.
 186. A method of modifyingfeeding behavior of a subject which comprises administering to thesubject an amount of a compound which is a GALR2 receptor agonist orantagonist effective to increase or decrease the consumption of food bythe subject so as to thereby modify feeding behavior of the subject.187. The method of claim 186, wherein the compound is a GALR2 receptorantagonist and the amount is effective to decrease the consumption offood by the subject.
 188. The method of claim 186 or 187, wherein thecompound is administered in combination with food.
 189. The method ofclaim 186, wherein the compound is a GALR2 receptor agonist and theamount is effective to increase the consumption of food by the subject.190. The method of claim 186 or 189, wherein the compound isadministered in combination with food.
 191. The method of claim 186 or189, wherein the compound binds selectively to a GALR2 receptor. 192.The method of claim 191, wherein the compound binds to the GALR2receptor with an affinity greater than ten-fold higher than the affinitywith which the compound binds to a GALR1 receptor.
 193. The method ofclaim 191, wherein the compound binds to the GALR2 receptor with anaffinity greater than ten-fold higher than the affinity with which thecompound binds to a GALR3 receptor.
 194. The method of claim 191,wherein the compound binds to the GALR2 receptor with an affinitygreater than one hundred-fold higher than the affinity with which thecompound binds to a GALR1 receptor.
 195. The method of claim 191,wherein the compound binds to the GALR2 receptor with an affinitygreater than one hundred-fold higher than the affinity with which thecompound binds to a GALR3 receptor.
 196. The method of claim 186,wherein the subject is a vertebrate, a mammal, a human or a canine. 197.A method for determining whether a compound is a GALR2 antagonist whichcomprises: (a) administering to an animal a GALR2 agonist and measuringthe amount of food intake in the animal; (b) administering to a secondanimal both the GALR2 agonist and the compound, and measuring the amountof food intake in the second animal; and (c) determining whether theamount of food intake is reduced in the presence of the compoundrelative to the amount of food intake in the absence of the compound, soas to thereby determine whether the compound is a GALR2 antagonist. 198.A method of screening a plurality of compounds to identify a compoundwhich is a GALR2 antagonist which comrises: (a) administering to ananimal a GALR2 agonist and measuring the amount of food intake in theanimal; (b) administering to a second animal the GALR2 agonist and atleast one compound of the plurality of compounds and measuring theamount of food intake in the animal; (c) determining whether the amountof food intake is reduced in the presence of at least one compound ofthe plurality relative to the amount of food intake in the absence of atleast one compound of the plurality, and if so; (d) separatelydetermining whether each compound is a GALR2 antagonist according to themethod of claim 132, so as to thereby identify a compound which is aGALR2 antagonist.
 199. The method of claim 197 or 198, wherein the GALR2agonist is [D-Trp]₂-galanin₍₁₋₂₉₎.
 200. The method of either of claims197 or 198 wherein the animal is a non-human mammal.
 201. The method ofclaim 200, wherein the mammal is a rodent.
 202. A process of claim 81 or82, which further comprises determining whether the compound selectivelybinds to the GALR2 receptor relative to another galanin receptor. 203.The process of claim 202, wherein the determination whether the compoundselectively binds to the GALR2 receptor comprises: (a) determining thebinding affinity of the compound for the GALR2 receptor and for suchother galanin receptor; and (b) comparing the binding affinities sodetermined, the presence of a higher binding affinity for the GALR2receptor than for such other galanin receptor inicating that thecompound selectively binds to the GALR2 receptor.
 204. A process ofclaim 202, wherein such other galanin receptor is a GALR1 receptor. 205.A process of claim 202, wherein such other galanin receptor is a GALR3receptor.
 206. A method of decreasing feeding behavior of a subjectwhich comprises administering a compound which is a GALR2 receptorantagonist and a compound which is a Y5 receptor antagonist, the amountof such antagonists being effective to decrease the feeding behavior ofthe subject.
 207. The method of claim 206, wherein the GALR2 antagonistand the Y5 antagonist are administered in combination.
 208. The methodof claim 206, wherein the GALR2 antagonist and the Y5 antagonist areadministered once.
 209. The method of claim 206, wherein the GALR2antagonist and the Y5 antagonist are administered separately.
 210. Themethod of claim 209, wherein the GALR2 antagonist and the Y5 antagonistare administered once.
 211. The method of claim 209, wherein the galaninreceptor antagonist is administered for about 1 week to 2 weeks. 212.The method of claim 209, wherein the Y5 receptor antagonist isadministered for about 1 week to 2 weeks.
 213. The method of claim 209,wherein the GALR2 antagonist and the Y5 antagonist are administeredalternately.
 214. The method of claim 213, wherein the GALR2 antagonistand the Y5 antagonist are administered repeatedly.
 215. A method ofclaim 213 or 214, wherein the galanin receptor antagonist isadministered for about 1 week to 2 weeks.
 216. A method of claim 213 or214, wherein the Y5 receptor antagonist is administered for about 1 weekto 2 weeks.
 217. A method of any one of claims 206, 207, 208, or 209,wherein the compound is administered in a pharmaceutical compositioncomprising a sustained release formulation.
 218. A method of decreasingnociception in a subject which comprises administering to the subject anamount of a compound which is a GALR2 receptor agonist effective todecrease nociception in the subject.
 219. A method of treating pain in asubject which comprises administering to the subject an amount of acompound which is a GALR2 receptor agonist effective to treat pain inthe subject.
 220. A method of treating Alzheimer's disease in a subjectwhich comprises administering to the subject an amount of a compoundwhich is a GALR2 receptor antagonist effective to treat Alzheimer'sdisease in the subject.